Recent advances in the application of core-shell structured magnetic materials for the separation and enrichment of proteins and peptides.

G Model

ARTICLE IN PRESS

CHROMA-355370; No. of Pages 12

Journal of Chromatography A, xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Recent advances in the application of core–shell structured magnetic materials for the separation and enrichment of proteins and peptides Man Zhao, Yiqin Xie, Chunhui Deng ∗ , Xiangmin Zhang Department of Chemistry, Fudan University, Shanghai 200433, China

a r t i c l e

i n f o

Article history: Received 11 March 2014 Received in revised form 20 April 2014 Accepted 22 April 2014 Available online xxx Keywords: Core–shell structure Magnetic microspheres Enrichment Low-abundance proteins/peptides Phosphoproteins Glycoproteins

a b s t r a c t Many endogenous proteins/peptides and proteins/peptides with post-translational modifications (PTMs) are presented at extremely low abundance, and they usually suffer strong interference with highly abundant proteins/peptides as well as other contaminants, resulting in low ionization efficiency in MS analysis. Therefore, the separation and enrichment of proteins/peptides from complex mixtures is of great importance to the successful identification of them. Core–shell structured magnetic microspheres have been widely used in the enrichment and isolation of proteins/peptides, thanks to unique properties such as strong magnetic responsiveness, outstanding binding capacity, excellent biocompatibility, robust mechanical strength and admirable recoverability. The aim of this review is to update the advances in the application of core–shell structured magnetic materials for proteomics analysis, including the separation and enrichment of low-concentration proteins/peptides, the selective enrichment of phosphoproteins and the selective enrichment of glycoproteins, and to compare the enrichment performance of magnetic microspheres with different kinds of functionalization. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Peptide mapping based on mass spectrometry (MS) along with database searching has become an indispensible tool in proteomics analysis [1–4], which is the premise of shotgun proteomics strategy. Though MS is highly sensitive to trace amount of proteins or peptides, it appears to be insufficient for the detection of low-concentration proteins/peptides that exist in real biological samples. Proteins/peptides extracted from biological samples are not only expressed at extremely low concentrations (less than 1 nM), but also suffer strong interference with highly abundant proteins/peptides as well as contaminants like buffer salts or surfactants that are introduced into the samples during pretreatment process [5]. Therefore, the enrichment of low-abundance peptides from complex mixtures is prior to MS analysis in most cases involving peptides identification. On the other hand, proteins/peptides with post-translational modifications (PTMs) have stimulated a tremendous amount of research interest in proteomics, since PTMs of a protein are involved in many cellular processes and can determine its activity state, localization, turnover and interactions with other proteins [6]. Furthermore, the aberrant modification of proteins may induce the

∗ Corresponding author. Tel.: +86 21 65643983; fax: +86 21 65641740. E-mail address: [email protected] (C. Deng).

dysfunction of cells, which is associated with certain human diseases [7]. However, effective analysis of these modified peptides with MS is still a challenging task because of their low abundance and low ionization efficiency than non-modified peptides [8]. Over the past years, core–shell structured magnetic materials, composed of an inorganic magnetic core and a functionalized shell (or a functionalized outer shell together with a hydrophilic intermediate shell), have been widely utilized in various sample preparation procedures thanks to their strong magnetic responsiveness, small diameters advantageous for high sensitivity, excellent biocompatibility, outstanding binding capacity and admirable recoverability [9]. Iron-oxide particles (Fe3 O4 and ␥Fe2 O3 ) are the most ubiquitous inner contents because they are easy to prepare and convenient to modify. Functionalized magnetic materials facilitate the isolation of nanomaterial–target molecule conjugates from sample solutions with the help of a magnetic field. After proper modifications, they may possess satisfactory dispersibility in aqueous solution. In a typical magnetic solid-phase extraction process, magnetic microspheres are dispersed in the sample solution at first. After incubation for an appropriate period of time until the target analytes are adsorbed on the adsorbent, the microspheres can easily be separated from the solution in an external magnetic field. Afterwards, the conjugates are properly rinsed and eluted, followed by MS analysis. The speciality of core–shell structured microspheres is the combined properties of each component which retains its identity and

http://dx.doi.org/10.1016/j.chroma.2014.04.078 0021-9673/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: M. Zhao, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.078

G Model CHROMA-355370; No. of Pages 12

ARTICLE IN PRESS M. Zhao et al. / J. Chromatogr. A xxx (2014) xxx–xxx

2

performs its function independently [10]. Multifunctional composite materials have shown improved enrichment efficiency in reduced time. In addition, the unique structure offers the possibility to accurately control the spatial distribution of the added species around the cores. Though physical adsorption is an alternative for modification, the most frequently used functionalization technique is definitely the formation of covalent bonds between the core and the shell. Covalent linkages endow composites with greater mechanical strength and stability enough for reuse, which is crucial to practical application [11,12]. This review mainly focuses on the present advances in the syntheses and applications of core–shell structured magnetic microspheres in sample preparation for proteomics analysis, including the enrichment of low-concentration proteins/peptides, the selective enrichment of phosphoproteins/phosphopeptides and the selective enrichment of glycoproteins/glycopeptides. 2. Enrichment of low-concentration proteins/peptides Endogenous peptides such as hormones, cytokines are shown to contain potential biomarkers for recording the physiological and pathological states of human beings. Unfortunately, most of them are low-concentration peptides [13,14]. Continuous efforts have been devoted to qualitative and quantitative study of endogenous peptides in biological environment by employing simple, rapid, convenient and universal protocols. Undoubtedly, concentration of low-abundance proteins/peptides from complex mixtures to inhibit the interference from unfavorable substances is necessary for successful MS analysis. In this section, advances in the application of core–shell structured magnetic materials for the enrichment of low-concentration proteins/peptides will be discussed. Core–shell structured magnetic microspheres for the enrichment of low-concentration proteins/peptides published in recent years are summarized in Table 1. 2.1. Hydrophobic materials Magnetic microspheres modified with n-alkyl chains have normally been used to enrich hydrophobic peptides through hydrophobic–hydrophobic interactions. 2.1.1. C8 -functionalized magnetic microspheres Among them, C8 -functionalized magnetic particles received the most wide-spread concern. Several kinds of C8 -functionalized magnetic microspheres prepared via different methods have been applied to the efficient enrichment of low-concentration peptides [15–19]. C8 -functionalized core–shell structured magnetic microspheres were synthesized for the first time via a conventional silanization method [15]. C8 chains were grafted onto the surface of silica-coated Fe3 O4 microspheres with the aid of n-octyldimethylchlorosilane. The resulting Fe3 O4 @nSiO2 @C8 microspheres displayed well-defined core–shell–shell structure and high dispersibility, and enriched hydrophobic peptides in the solutions of a standard peptide Angiotensin II, bovine serum albumin (BSA) and myoglobin (MYO) tryptic digest, and human serum. The enrichment factor was estimated to be nearly 100 times. Twenty-six peptides with sequence coverage of 42% could be identified for 5 nM BSA tryptic digest, and 11 peptides with sequence coverage of 79% could be identified for 5 nM MYO digest after treatment with Fe3 O4 @nSiO2 @C8 . Additionally, many endogenous peptides in human serum came into detection after enrichment with Fe3 O4 @nSiO2 @C8 . Considering the synthetic route mentioned above was time-consuming and low-yield, C8 -functionalized magnetic microspheres were synthesized by a one-pot process where Fe3 O4 microspheres were modified with amine and chloro(dimethyl)

octylsilane in succession [16]. After being enriched by Fe3 O4 –NH2 @C8 microspheres for only 30 s, matched peptides with high intensity dominated the MS spectra for 5 nM MYO digest. The enrichment approach based on Fe3 O4 –NH2 @C8 also showed good reproducibility and a detection limit of 20 nM. Another option to avoid the complex silica coating process was to coat Fe3 O4 nanoparticles with carbonaceous polysaccharide [17]. The as-synthesized Fe3 O4 @CP@C8 microspheres could identify 22 peaks with sequence coverage of 35% in 5 nM BSA tryptic digest solution which contained abundant contaminants (100 mM urea), thus obviating the need for a desalting step. The feasibility of employing Fe3 O4 @CP@C8 composites in real proteomic applications was further confirmed by direct analysis of peptides present in the digestion mixture of a protein spot that was obtained via 2D-PAGE analysis of human-eye lens. The protein identified with Fe3 O4 @CP@C8 got a score of 96. In all of the above-mentioned reports, C8 -functionalized magnetic microspheres suffered from limited specific area. Hence, silica-coated magnetic microspheres were fabricated with an ordered mesoporous hybrid C8 layer to settle this issue. The asprepared Fe3 O4 @nSiO2 @meso-hybrid-C8 microspheres possessed high surface area and large pore volume, and achieved better performance than Fe3 O4 –NH2 @C8 when treating BSA tryptic digests [18]. The detection of limit (LOD) for BSA digest was 1 nM. Detectable peptides in a tryptic digest of rat cerebellum proteins increased in number from 3 to 15, and numerous endogenous peptides in human serum were successfully detected after pretreatment with Fe3 O4 @nSiO2 @meso-hybrid-C8 . However, the fabrication of Fe3 O4 @nSiO2 @meso-hybrid-C8 microspheres still involved the introduction of silica layer. C8 -functionalized magnetic mesoporous silica (designated as Fe3 O4 @mSiO2 @C8 ) microspheres preserved the merits of distinguished surface area and pore volume and avoided the time-consuming silica coating. The enrichment efficiency of the novel material was superior to that of Fe3 O4 @nSiO2 @C8 [19]. Originated from the unique mesoporous structure, Fe3 O4 @mSiO2 @C8 could selectively capture proteins with low molecular weight (MW) and exclude BSA and other large proteins simultaneously. Fe3 O4 @mSiO2 @C8 microspheres were also demonstrated to be capable of capturing endogenous peptides from human serum and mouse brain extract. Notably, 267 peptides were finally identified from mouse brain extract. 2.1.2. Other carbon-based magnetic microspheres Other carbon-based composites like Fullerene (C60)functionalized magnetic microspheres (Fe3 O4 @nSiO2 @C60), carbon nanotube (CNT)-decorated magnetic microspheres and graphene-encapsulated magnetic microspheres (Fe3 O4 @nSiO2 @G) were demonstrated to have extraordinary capability for the enrichment of low-concentration peptides thanks to the strong hydrophobic interactions between the carbon skeletons and peptides [11,20,21]. Fe3 O4 @nSiO2 @C60 microspheres were synthesized by radical polymerization of C60 molecules on the surface of Fe3 O4 microspheres. C60 was anchored onto the surface of Fe3 O4 @nSiO2 by the use of 3-(trimethoxysilyl) propylmethacrylate (MPS), and the MPS-modified Fe3 O4 @nSiO2 was then polymerized with C60 by using 2,2-azobisiso-butyronitrile (AIBN) as the polymerization initiator [20]. The enrichment factor for Angiotensin II was estimated to be over 100 times and the enrichment yield was about 0.12 ng peptide per microgramme of Fe3 O4 @nSiO2 @C60 microspheres. The material not only remarkably concentrated peptides in BSA tryptic digest solutions, but also proved to be effective for the enrichment of Cytochrome c (Cytc) protein at a concentration of 0.2 ng/␮L. What is more, Fe3 O4 @nSiO2 @C60 could also directly analyze human urine sample without additional desalting step.


Hydrophobic materials

C8 -functionalized magnetic microspheres

Sensitivity

Enrichment factor

Tryptic digest

Real samples

Fe3 O4 @nSiO2 @C8

Lower than 5 nM

About 100

Human serum

Fe3 O4 -NH2 @C8

20 nM in human serum Lower than 5 nM

Not mentioned

5 nM BSA (26 peptides), 5 nM MYO (11 peptides) 5 nM MYO

54.5

5 nM BSA with 100 mM urea (22 peptides)

1 nM for BSA tryptic digest Lower than 5 nM

41.4 at most

15 nM BSA (13 peptides)

About 34

0.5 nM and 0.2 ng/␮L Cytc 0.6 nM

Over 100 Not mentioned

5 nM BSA (26 peptides), 5 nM MYO (11 peptides) 1 nM BSA with 100 mM CaCl2 (15 peptides), –

304

7.6 nM BSA (7 peptides)

In vitro selection of ssDNA aptamers against carcinoembryonic antigen Human saliva

Fe3 O4 @CP@C8

Fe3 O4 @mSiO2 @C8 Other carbon-based magnetic microspheres

Fe3 O4 @nSiO2 @C60 Fe3 O4 @CNT

Fe3 O4 @nSiO2 @G

Human serum The digestion mixture of a protein spot obtained via 2D-PAGE analysis of human-eye lens Human serum, rat cerebellum proteins (15 peptides) Human serum, mouse brain extract (267 peptides) Human urine

Not mentioned

5 nM BSA (19 peptides)

–

Fe3 O4 @OA Fe3 O4 @nSiO2 @PMMA

7.6 nM for BSA tryptic digest, standard proteins Lower than 5 nM, standard proteins 0.2 nM Lower than 2 nM

51.6 60.4

5 nM MYO (14 peptides) 2 nM BSA with 100 mM urea (14 peptides)

Human serum Proteins extracted from human eye lens

Fe3 O4 @nSiO2 @mSiO2

Lower than 5 nM

Over 100

Rat brain extract

Fe3 O4 @mSiO2

Lower than 5 nM, proteins with low MW

62.1

5 nM MYO (15 peptides), 5 nM BSA (19 peptides) 10 nM BSA with 1 M NaCl (20 peptides), 10 nM BSA with 1 M urea (21 peptides)

IMAC-based materials

Fe3 O4 @mSiO2 -Cu2+ Fe3 O4 @[Cu3 (btc)2 ]

Lower than 5 nM 10 pM

40.4 13

5 nM BSA (18 peptides) 10 nM BSA (7 peptides), 10 nM MYO (15 peptides)

Human serum Human urine

Materials with other modifications

Isothiocyanate-functionalized Fe3 O4 @nSiO2

10 pmol for kemptide

1.58

BSA, MYO (15 peptides)

Hydrazide-functionalized Fe3 O4

Selectivity 1:100

1860

–

Fe3 O4 @CP@Au@aptamer

0.36 nM thrombin

Not mentioned

–

Tryptic digest of protein extract from HepG2 cells (215 N-terminal peptides) A crude fraction of mouse serum with ovarian cancer Human serum

Fe3 O4 @G Magnetic microspheres with other derivatives Mesoporous materials

Human plasma

ARTICLE IN PRESS

Fe3 O4 @nSiO2 @meso-hybrid-C8

G Model

CHROMA-355370; No. of Pages 12

Type of core–shell structured microspheres

M. Zhao et al. / J. Chromatogr. A xxx (2014) xxx–xxx


Table 1 Core–shell structured magnetic microspheres for the enrichment of low-concentration proteins/peptides.

3

G Model CHROMA-355370; No. of Pages 12 4


Fig. 1. Schematic illustration of the fast and facile enrichment protocol for endogenous peptides by using Fe3 O4 @nSiO2 @mSiO2 microspheres [28].

Fe3 O4 @CNT microspheres could effectively capture DNA oligonucleotides through strong physical wrapping of DNA around CNTs’ wall [21] and were likely to be a potential platform in Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. To prepare Fe3 O4 @nSiO2 @G, silica-coated Fe3 O4 microspheres were functionalized with (3-aminopropyl)triethoxysilane (APTES) in advance to turn into positively charged. Subsequently, negatively charged GO sheets synthesized via Hummers method were assembled on amino-functionalized silica-coated Fe3 O4 through electrostatic interactions. Eventually, GO sheets were reduced to G with hydrazine [11]. After treatment with Fe3 O4 @nSiO2 @G, the S/N ratios of peaks in MYO and BSA triyptic digest solutions increased 2 orders of magnitude. Fe3 O4 @nSiO2 @G was also capable of enriching standard proteins via hydrogen bonding and electrostatic interaction, and recoveries of the proteins on Fe3 O4 @nSiO2 @G ranged from 58.2 to 101.2%, and the LOD ranged from 3.8 to 68 fmol. The enrichment efficiency was superior to that of commercial C18 adsorbent. Another way to prepare Fe3 O4 @GO and Fe3 O4 @G composites was to treat Fe3 O4 with 0.1 M hydrochloric acid beforehand and to add the particle suspension dropwise into the ethanol solution of MPSmodified GO [22]. The adsorption capacity of the proteins adsorbed by Fe3 O4 @GO microspheres was as high as 294.54 mg/g. Nineteen peptides with sequence coverage of 21% could be assigned to BSA with enhanced intensities for BSA tryptic digest solution at a concentration of 5 nM. 2.1.3. Magnetic microspheres with other derivatives A variety of functionalized magnetic microspheres with derivatives other than carbon-based composites were designed to enrich low-concentration proteins/peptides from biological samples. Oleic acid-functionalized magnetic microspheres (denoted as Fe3 O4 @OA) with average diameter of 15 nm can be synthesized through a one-pot process [23]. The limit of detection (LOD) obtained with Fe3 O4 @OA was 0.2 nM for Angiotensin II, lower than that of C8 -functionalized magnetic microspheres. The results might be ascribed to the higher hydrophobic property and the unique nano size of Fe3 O4 @OA. Poly(methyl methacrylate) (PMMA), an organic hydrophobic polymer, was also revealed to be a powerful adsorbent for the enrichment of peptides and proteins due to linear hydrophobic chains [24]. With a modified method based on the abovementioned work, PMMA was functionalized on MPS-modified Fe3 O4 @nSiO2 microspheres by initiating an aqueous-phase radical polymerization of methyl methacrylate (MMA) [25]. After

enrichment with Fe3 O4 @nSiO2 @PMMA, the S/N ratio of Cytc at a concentration of 0.5 mg/L got sharp increase, since the Fe3 O4 @nSiO2 cores served to block target molecules, effectively constraining them to the thin outer layers of the microspheres. Fifteen peptides with sequence coverage of 24% for 2 nM BSA digest and 14 peptides with sequence coverage of 22% for 2 nM BSA digest with 100 mm urea could be determined, indicating effective enrichment for both samples regardless of contamination by urea. 2.2. Mesoporous materials Mesoporous materials with tunable pore sizes are promising candidates to enrich proteins or peptides with different MWs based on size-exclusion mechanism. Especially in the treatment of low-abundance peptides, the application of mesoporous materials can effectively suppress the interference with high-abundance proteins due to the fact that peptides are retained inside the pores by weak hydrophobic interactions with the siloxane bridge groups [26,27]. Microspheres composed of Fe3 O4 @nSiO2 cores and perpendicularly aligned mesoporous SiO2 shells (designated as Fe3 O4 @nSiO2 @mSiO2 ) [28] were characterized by highly ordered nanoscale pores (2 nm) and high surface area. An increase in S/N ratio of over 100 times could be achieved for Angiotensin II after enrichment with Fe3 O4 @nSiO2 @mSiO2 microspheres, slightly better than the results obtained with Fe3 O4 @nSiO2 @C8 . Fifteen assignable peptides with sequence coverage of 93% were identified for a MYO tryptic digest solution at the concentration of 5 nM and 19 matched peptides with sequence coverage of 32% were identified for a BSA tryptic digest solution at the concentration of 5 nM after treatment. More importantly, large-scale enrichment of endogenous peptides in rat brain extract was achieved by the material, with 60 unique peptides identified by one-step eluted fractions. The workflow for peptidome enrichment is illustrated in Fig. 1. Another kind of mesoporous silica microspheres (Fe3 O4 @mSiO2 ) were obtained through pseudomorphic synthesis to transform non-porous magnetic silica (Fe3 O4 @nSiO2 ) into ordered Fe3 O4 @mSiO2 [29]. The nanocomposites exhibited a narrow pore size distribution curve, which centered at the mean value of 2.9 nm. Twenty matched peptides were detected for 10 nM BSA tryptic digest after enrichment with Fe3 O4 @mSiO2 . Proteins with low MW could also be enriched in spite of the interference with high-MW proteins. Fe3 O4 @mSiO2 could also enrich peptides in human plasma with high desalting efficiency.




2.3. Immobilized metal ion affinity chromatography (IMAC)-based materials For porous silica particles, peptides are retained by the weak hydrophobic interaction with the siloxane-bridge groups on the surface of inner pores. However, many endogenous peptides are hydrophilic, and may be lost because of the weak hydrophobic interactions. Cu2+ ions can be immobilized on substrates and interact with amino acids’ chains of the proteins/peptides through coordination bond, and after enrichment the protein/peptide can be released through the disruption of coordination bond using eluting reagent. Therefore, Cu2+ -immobilized magnetic mesoporous silica microspheres (denoted as Fe3 O4 @mSiO2 –Cu2+ ) were synthesized to enrich endogenous peptides with high hydrophilicity [30]. The high surface area and large amount of immobilized Cu2+ ions enabled Fe3 O4 @mSiO2 –Cu2+ microspheres to enrich both hydrophilic and hydrophobic peptides by chelating with carboxylic and amino groups of peptides. By applying the IMAC-based magnetic adsorbent, the enrichment factor for hydrophilic standard peptide microcystin-LR (MC-LR) was 35 and the enrichment factor for hydrophobic Angiotensin II was 40. In the case of BSA tryptic digest, Fe3 O4 @mSiO2 –Cu2+ microspheres enriched hydrophilic and hydrophobic peptides ranging in grand average of hydropathy (GRAVY) from −1.723 to 0.292 without discrimination. Core–shell structured magnetic metal organic framework (MOF) microspheres Fe3 O4 @[Cu3 (btc)2 ] were synthesized by coating Fe3 O4 particles with mercaptoacetic acid and subsequent reactions with ethanol solutions of Cu(OAc)2 and benzene-1,3,5-tricarboxylic acid (designated as H3 btc) alternately [31]. After treatment with the novel magnetic MOF microspheres, 15 peptides in a 10 nM MYO tryptic digest solution could be assigned to MYO with sequence coverage up to 89%. Fe3 O4 @[Cu3 (btc)2 ] microspheres also showed higher enrichment capacity for peptides in standard protein trytic digest than Fe3 O4 @nSiO2 @C8 and Fe3 O4 @CP@C8 .

5

hydrophobic materials was 0.2 nM and the greatest enrichment factor was 304 times. Mesoporous materials can effectively suppress the interference with high-abundance proteins, with the lowest LOD of 5 nM and the greatest enrichment factor of over 100 times. IMAC-based materials can enrich both hydrophobic and hydrophilic peptides without discrimination, with the lowest LOD of 10 pM and the greatest enrichment factor of 40.4. Materials with other functionalizations are favorable to certain proteins or peptides containing terminal or specific amino acids. And all of the above-mentioned kinds of magnetic materials were demonstrated to be effective in the treatment of practical samples such as human serum, human urine, rat brain extract, mouse brain extract, etc.

3. Enrichment of phosphoproteins/phosphopeptides Phosphorylation is one of the most ubiquitous posttranslational protein modifications in living cells, which plays a vital role in signal-transduction, gene expression, metabolism, cell growth, division and differentiation [35,36]. It is estimated that 30–50% of all proteins in a cell are phosphorylated at any given time [37,38], so investigation into phosphorylation are of keen interest in the field of proteomics. However, phosphopeptides are often presented at low abundance [39,40] and phosphorylation at individual sites in a protein is usually incomplete [41,42]. Therefore, selective enrichment of phosphorylated peptides is a prerequisite to MS analysis. To meet this requirement, various methods have been advanced to selectively separate phosphorylated proteins and peptides from nonphosphorylated ones such as IMAC [43,44], metal oxide affinity chromatography (MOAC) [45–47], immunoprecipitation [48], strong cation-exchange chromatography [49], etc. The typical procedure to enrich phosphopeptides from a tryptic digest of standard proteins or practical samples is displayed in Fig. 2.

2.4. Materials with other modifications 3.1. Immobilized metal ion affinity chromatography (IMAC) In addition to the enrichment techniques based on hydrophobic interactions or coordination binding listed above, some methods concentrated on the selective enrichment of certain proteins or peptides containing terminal or specific amino acids. For instance, isothiocyanate-coupled magnetic microspheres were developed to isolate and identify N-blocked peptides from tryptic digest of protein extract of HepG2 cells, combined with preenrichment by strong cation exchange (SCX) chromatography [32]. Hydrazide-functionalized magnetic microspheres were exploited to selectively enrich tryptophan (Trp)-containing peptides from complex samples with low volume [33]. Furthermore, combined with 1D-LC–MS/MS analysis, the strategy was successfully applied to the proteomic study of mouse serum, with an increase in the proportion of Trp-containing peptides from 19.4% to 80.2%. Additional 113 Trp-containing peptides and 48 novel proteins were detected compared with conventional methods. Thrombin binding aptamer-functionalized Fe3 O4 @CP@Au microspheres were used to extract trace level of thrombin from dilute solutions [34]. The LOD achieved by this approach was as low as 18 fmol, corresponding to 0.36 nM thrombin in 50 ␮L of original solution. Linear relation was observed within a concentration range from 0.5 nM to 10 nM with linear correlation R2 = 0.998. Other proteins including human serum albumin (HSA), Ig G, transferrin, oval albumin (OVA) and fetal calf serum did not interfere with thrombin detection. To sum up, among all kinds of core–shell structured magnetic materials designed for the enrichment of low-concentration proteins/peptides, hydrophobic materials tend to enrich hydrophobic peptides with high selectivity. The lowest LOD achieved by

The most commonly adopted strategy is IMAC which relies on the affinity of phosphate moiety toward metal ions (such as Fe3+ , Ti4+ , Zr4+ ) immobilized on different adsorbents. IMAC-based method is fast, easy-to-handle and economical, and the preparation of magnetic IMAC materials was easier than materials based on other protocols. Fe3+ -immobilized magnetic microspheres were synthesized as the first example of core–shell structured magnetic microspheres to extract phosphopeptides form tryptic digest of ␤-casein through direct MALDI-TOF MS analysis [50]. Fe3 O4 @nSiO2 microspheres were firstly functionalized with a silane coupling agent (GLYMO-IDA) derived from the reaction of 3glycidoxypropyltrimethoxysilane (GLYMO) with iminodiacetic acid (IDA), and then with Fe3+ ions by chelation. Two phosphopeptides and one dephosphorylated fragment could be identified for 0.2 nmol/mL ␤-casein digest, and peptides adsorbed on the Fe3+ -immobilized Fe3 O4 @nSiO2 microspheres could be directly analyzed without elution. Ce4+ -immobilized Fe3 O4 @nSiO2 microspheres were synthesized by employing the same chelating ligand [51]. Ce4+ -immobilized Fe3 O4 @nSiO2 had higher affinity and better selectivity toward phosphopeptides than the former material in treating tryptic digest of mixed peptides, tryptic digest of ␤casein and BSA with a molar ratio of 1:50 and tryptic digest of nonfat milk samples. Eight peaks derived from ␣-casein, two peaks derived from ␤-casein and several dephosphorylated fragments were observed after treatment with the Ce4+ -based composites. Besides, three phosphopeptides in human serum were rapidly enriched with high intensities by Ce4+ -immobilized Fe3 O4 @nSiO2 .




Fig. 2. Workflow of phosphopeptides enrichment from a biological sample using core–shell structured magnetic microspheres [57].

However, it was evaluated that using NTA as the chelating ligand provided better phosphopeptide selectivity and yield than using IDA [52]. Thus, Zr4+ and Ga3+ immobilized Fe3 O4 @nSiO2 NTA nanoparticles were prepared to enrich phosphorylated peptides [53]. Both Zr4+ -immobilized Fe3 O4 @nSiO2 -NTA and Ga3+ -immobilized Fe3 O4 @nSiO2 -NTA microspheres displayed high affinity toward phosphopeptides with the LOD of 50 fmol for tryptic digests of ␣-casein and ␤-casein. Enrichment was achieved through vigorously mixing the sample solution and the nanoparticles by pipetting in and out of a sample vial for only 30 s. The protein mixtures were also characterized by enrichment on the Zr4+ /Ga3+ -immobilized Fe3 O4 @nSiO2 -NTA affinity probes using onprobe tryptic digestion under microwave irradiation for only 2 min [54,55]. In order to reduce the adsorption of nonphosphorylated peptides for IMAC-based methods, IDA-modified magnetic hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [poly(HEMA-GMA)] microspheres were prepared by dispersion copolymerization of HEMA and GMA in the presence of Fe3 O4 , followed by modification with IDA [56]. The products were further immobilized with Fe3+ or Ga3+ ions after being washed with 0.1 M FeCl3 or GaCl3 solution. Due to the hydrophilicity of HEMA, the magnetic polymer can effectively suppress undesirable non-specific adsorption. Only single phosphopeptide in Porcine pepsin A, which contains a relatively high number of acidic amino acid residues that limit phosphopeptide separation procedures, was recovered after enrichment with Fe3+ /Ga3+ -immobilized Fe3 O4 @[p(HEMA-GMA)]-IDA. Polydopamine (PDA)-modified Fe3 O4 microspheres were considered to be a new kind of substrates to immobilize metal ions. Ti4+ -immobilized Fe3 O4 @PDA microspheres were synthesized to selectively enrich phosphopeptides from ␤-casein tryptic digest solutions and human serum [57]. A LOD of 2 fmol and a selectivity of 500-fold dilution by BSA tryptic digest were achieved for ␤-casein tryptic digest with the PDA-based IMAC material. Considering the marvelous dispersibility, favorable biocompatibility and facile formation of polydopamine, PDA-coating strategy is likely to be an ideal candidate for the immobilization of metal ions in IMAC technique.

3.2. Phosphate/arsenate-based IMAC Recently, a new kind of IMAC adsorbent – zirconium phosphonate-modified porous silicon was employed for the enrichment of phosphopeptides [58]. In 2008, zirconium phosphonatefunctionalized Fe3 O4 @nSiO2 microspheres were synthesized for phosphoproteome study [59,60]. Under optimized experimental conditions, 10−9 M phosphopeptides in 50 ␮L tryptic digest of ␤-casein could be enriched and identified, suggesting a LOD of fmol level. Only 4 phosphopeptide peaks originated from ␤casein were presented in MS spectra because of the elimination of ion suppression from nonphosphorylated peptides and salts after selective capture of phosphopeptides using Zr4+ -immobilized Fe3 O4 @nSiO2 –PO3 . Phosphopeptides in a tryptic digest solution of ␤-casein and BSA with the molar ratio of 1:100 could successfully be isolated. The application feasibility of the zirconium phosphonatecoated Fe3 O4 microspheres in real samples was verified by a tryptic digest of a protein extract from Chang liver cells and the phosphoproteome of mouse liver. After that, Zr4+ -immobilized Fe3 O4 @CP–PO3 microspheres were synthesized for the selective extraction of phosphopeptides from ␤-casein and achieved a LOD of 2 fmol [61]. Reversed phase liquid chromatography (RPLC) was employed to investigate the quantitative recovery of phosphopeptides/nonphosphopeptides of the core–shell structured microspheres. It was discovered that the recovery of two phosphopeptides was estimated to be 86.3% and 93.4%, respectively, while the recovery of a nonphosphopeptide was calculated to be merely 0.6%, exhibiting marvelous selectivity. Nonfat bovine milk and tryptic digest of rat brain were used to evaluate the performance of Zr4+ -immobilized Fe3 O4 @CP–PO3 in practical environment. In result, 192 phosphorylation sites–164 on serine (85.42%), 27 on threonine (14.06%) and one on tyrosine (0.52%) were identified. Another zirconium phosphonate-based material, Zr4+ immobilized Fe3 O4 @poly(VPA-EDMA-x)–PO3 magnetic polymer microspheres were designed for the same purpose [62]. By exploring the relationship of enrichment efficiency with the number of chelating agent, the researchers found that the capturing ability of the material was related to the phosphate group content. When




the attached phosphate group content was low, only limited chelating sites were available for immobilizing Zr4+ , leading to poor enrichment efficiency; however, if the content of phosphate groups was too high, there would be an excess of tridentate anionic ligands sharing oxygen atoms with metal ions. With an optimized content of chelating agent, they successfully identified 988 unique phosphopeptides and 1276 phosphorylation sites from a proteolytic digest of HeLa cell extracts. Later on, Zr4+ -immobilized Fe3 O4 @mSiO2 –PO3 microspheres were fabricated to improve the surface area of phosphate-based magnetic IMAC materials [63]. The LOD for ␤-casein tryptic digest decreased to 0.2 fmol by virtue of mesoporous silica shells. The novel material also showed good reusability, and identified 218 phosphorylation sites - 175 on serine (80.27%), 38 on threonine (17.43%) and 5 on tyrosine (2.29%) from tryptic digest of rat brain. A new type of Ti4+ -immobilized magnetic IMAC material, with adenosine triphosphate (ATP) as the chelating ligand was synthesized for the enrichment of phosphopeptides [64]. For the first time, the approach for phosphopeptide enrichment provided selectivity under 5000-fold dilution by nonphosphopeptides, and a sensitivity of on-plate enrichment at 3 amol. The improved specificity and sensitivity was probably because the ATP molecule, which is composed of three phosphate groups, could offer superiorly strong and active metal phosphonate sites to bind phosphopeptides. Moreover, its hydrophilic purine base and pentose sugar groups might contribute great hydrophilicity to inhibit the non-specific adsorption of non-phosphopeptides. By the utilization of Ti4+ -immobilized Fe3 O4 –NH2 –ATP microspheres, 406 unique phosphopeptides with 538 phosphorylation sites in rat liver mitochondria, corresponding to 313 phosphoprotein groups, were identified. A chelating ligand containing arsenate groups was also employed to modify magnetic microspheres for Zr4+ chelation and phosphopeptide enrichment [65]. A higher selectivity for the specific capture of phosphopeptides and a better capture capability toward multiply-phosphorylated peptides from complex tryptic digests were achieved by Zr4+ -immobilized Fe3 O4 @nSiO2 –AsO3 than commercial zirconium dioxide (ZrO2 ). A LOD of 10 fmol was obtained with no interference from nonphosphopeptides. 3.3. Metal oxide affinity chromatography (MOAC) During the past decades, metal oxide affinity chromatography (MOAC) has gained increasing attention. MOAC has higher selectivity for phosphopeptides than IMAC because of their reduced nonspecific binding. On the other hand, traditional IMAC is severely affected by various buffers, detergents and other low molecular weight molecules. However, metal oxide is more robust and tolerant toward many reagents normally utilized in biochemical and cell biological procedures and the results are more reproducible. Varieties of metal oxides (TiO2 [66–76], ZrO2 [55,77], Al2 O3 [78,79], Ga2 O3 [80], SnO2 [81], ZnO [82], Ta2 O5 [83,84], Nb2 O5 [85] and CeO2 [86,87]) were successfully coated onto Fe3 O4 microspheres by using various synthesis protocols and were applied to the enrichment of phosphopeptides. Among these magnetic MOAC materials, TiO2 -immobilized Fe3 O4 microspheres have drawn most extensive attraction owing to their outstanding enrichment performance and stability. Before immobilization with TiO2 shells, Fe3 O4 microspheres were usually modified with an intermediate layer to increase the dispersibility or specific area of the composites, such as nSiO2 [68,70], mSiO2 [66,75], mesoporous hollow silica [70], carbonaceous polysaccharide [69], graphite [71], graphene oxide [72], poly(acrylic acid) [73] and mesoporous TiO2 with anatase form [74]. With increased specific area and dispersity, trapping capacity can be improved and time required for enrichment can be saved. Only a few works used bare Fe3 O4 microspheres for phosphopeptide enrichment without any coating [88,89]. The enrichment

7

process utilized affinity interactions between iron ions and phosphate groups. Meanwhile, it was unveiled that TiO2 with rutile form exhibited higher selectivity than the commonly used TiO2 with anatase form [90]. However, the application of TiO2 with rutile form was restricted for the sake of worse adsorption ability. To address this issue, magnetic microspheres with a rutile TiO2 nanorod shell were synthesized and put into use in phosphopeptides enrichment [76]. The echinus-like microspheres displayed stronger selectivity toward phosphopeptides than TiO2 with anatase form in the pretreatment of ␣-casein tryptic digest. And the microspheres could effectively eliminate the interference from high-abundance phosphocholines and lysophosphocholines in human plasma and improve the limit of detection for low-abundance metabolites. In addition, TiO2 -immobilized magnetic microspheres could be directly mixed with matrix and injected to MS analyzer without the elution step [67]. ZrO2 and Al2 O3 were also frequently used to modify magnetic microspheres for phosphopeptides enrichment, and it was reported that ZrO2 provided stronger affinity to monophosphorylated peptides than TiO2 [91], which is more sensitive to multiphosphorylated peptides. Al2 O3 also exhibited higher selectivity toward phosphorylated peptides than TiO2 [78,79]. It was found that Fe3 O4 @Ta2 O5 and Fe3 O4 @Al2 O3 had similar trapping specificity for phosphopeptides, but both of their enrichment selectivity were better than that of Fe3 O4 @TiO2 [83]. The universal synthetic route for core–shell structured metaloxide immobilized magnetic microspheres has already been summarized in a previous review [9]. In brief, Fe3 O4 microspheres were synthesized via a conventional solvothermal reaction at first. The resulting particles were then coated with a layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction. Subsequently, metal alkoxide was prehydrolyzed and adsorbed onto the microspheres through a sol–gel process and was finally converted into metal oxide through calcination. Illustration of the above-mentioned synthetic route is shown in Fig. 3. However, different from TiO2 , ZrO2 , Al2 O3 and Ga2 O3 , which can be successfully coated on Fe3 O4 microspheres using the regular strategy, tin dioxide (TiO2 ) cannot be generated on Fe3 O4 microspheres since TiO2 tends to crystallize during the sol-gel process. Fe3 O4 @CP@SnO2 microspheres were synthesized by a facile approach based on solvothermal and hydrothermal reactions [81]. Fe3 O4 @CP microspheres were subject to a solvothermal reaction with stannate trihydrate (K2 SnO3 ·3H2 O) in an ethanol/water mixture with urea as a promoter, resulting in SnO2 -immobilized magnetic microspheres. The LOD for ␤-casein was 2 fmol, comparable to many of the previous methods [68,77,78,80]. The preparation of CeO2 -immobilized magnetic microspheres is also notable. Mesoporous CeO2 shell was coated on magnetic microspheres via chemical precipitation and subsequent calcination process on the surface of Fe3 O4 @nSiO2 [86]. The mCeO2 shell avoided the ‘shadow effect’ which would hamper the release of phosphopeptides [25,92]. In addition to the ability to selectively enrich phosphopeptides, Fe3 O4 @nSiO2 @mCeO2 microspheres could also simultaneously catalyze the dephosphorylation of the enriched phosphopeptides, thereby producing labeling signals in mass spectra. The labeling capability of Fe3 O4 @nSiO2 @mCeO2 microspheres was tested with tryptic digest solutions of ␤-casein and BSA mixture and nonfat bovine milk. Significantly, the unique synthetic approach threw light on the preparation of rare-earth oxides with magnetic cores. 3.4. Immunoprecipitation and strong anion exchange (SAX) Core–shell structured microspheres were also employed in immunoprecipitation and SAX methods. Fe3 O4 beads coupled with anti-phosphotyrosine antibody 4G10 were designed to selectively




8

Fig. 3. The general synthetic route of metal-oxide immobilized magnetic microspheres [69].

enrich phosphotyrosine peptides as an example for immunoprecipitation [93]. The novel composite successfully enriched phosphotyrosine peptides mixed with non-phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were then used to enrich tyrosine phosphopeptides from a digest of the in vitro-phosphorylated recombinant P-intracellular region of the granulocyte-macrophage colony-stimulating factor receptor and were proved to be sensitive for tyrosine phosphoproteome analysis. As for SAX, polyethylenimine-modified MNPs (denoted as Fe3 O4 @PEI) were synthesized to concentrate phosphopeptides [94]. Plenty of amino groups on Fe3 O4 @PEI interacted strongly with phosphate groups on phosphopeptides, leading to extremely high specificity for phosphopeptides. By changing the composition of the binding solution, Fe3 O4 @PEI was able to convert their selectivity toward mono-phosphopeptides or multi-phosphopeptides, and they were successfully used to isolate phosphopeptides from a tryptic digest of nonfat milk. In summary, among all kinds of core–shell structured magnetic materials aimed at enriching phosphoproteins and phosphopeptides, materials based on traditional IMAC technique are distinguished by facile preparation, easy operation and economical raw materials. IMAC materials provided the lowest LOD of 2 fmol and the best selectivity under 500-fold dilution by nonphosphopeptides. Phosphate/arsenate-based IMAC materials have the advantage of abundant metal phosphonate sites to bind phosphopeptides, with the lowest LOD of 3 amol and the best selectivity under 5000-fold dilution by nonphosphopeptides. MOAC materials can effectively suppress the nonspecific binding with nonphosphopeptides, with the lowest LOD of 2 fmol and the best selectivity under 1000-fold dilution by nonphosphopeptides. Immunoprecipitation-based materials are able to enrich phosphotyrosine peptides mixed with non-phosphorylated peptides at a molar ratio of up to 1:200 and SAX-based materials can convert their selectivity toward mono-phosphopeptides or multiphosphopeptides. The feasibility of these materials in practical applications was also tested with human serum, rat brain extract, cell extracts, etc. 4. Enrichment of glycoproteins/glycopeptides Glycosylation is another common PTMs, which is critical to a series of biological processes (e.g., protein trafficking, folding, and molecular recognition) [7]. Similar to phosphopeptides, the ionization efficiency of glycopeptides is often suppressed by the co-existence of non-glycosylated peptides [9]. General

glyco-specific enrichment strategies include lectin-based affinity chromatography, hydrazide chemical method, boronate affinity chromatography and hydrophilic interaction chromatography (HILIC) [95,96]. The schemes of the above strategies for glycoproteins/glycopeptides enrichment are shown in Fig. 4.

4.1. Lectin-based affinity chromatography Lectins, a group of proteins with unique affinity toward specific glycan moieties, display a high degree of specificity for glycoproteins/glycopeptides [9]. Lectins have long been used in the isolation of glycoproteins/glycopeptides since the 1980s [97]. Concanavalin A (Con A) is one of the most frequently used lectins. Con A specifically binds mannosyl and glucosyl residues of polysaccharides and glycoproteins containing free hydroxyl groups at positions C3, C4 and C6 [98] and can be applied as a major tool for capturing N-glycosylated peptides and proteins with broad specificity [99]. Con A-immobilized magnetic microspheres were prepared for the first time by forming boronic acid-sugar-Con A bond in sandwich structure using methyl ␣-d-mannopyranoside as an intermedium [100]. The selectivity of Con A-immobilized aminophenylboronic acid (APBA)-modified Fe3 O4 for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocellular carcinoma cell line 7703. In consequence, 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Moreover, the regeneration of the Con A-modified MNPs can easily be performed, taking advantage of the reversible binding mechanism between the boronic acid and the sugar chain. Con A-conjugated magnetic microspheres were also employed to isolate the special membrane glycoproteins from living HepG-2 cells [101]. The isolated glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography–tandem mass (LC–MS/MS) spectrometry. A total of 37 membrane glycoproteins were identified, and 25 of them were ascertained to locate in the extracellular region. More recently, a high-throughput glycoprotein platform based on lectin-coupled magnetic beads (Dynabeads) was established to detect alterations in protein glycosylation [102]. Isolated glycoproteins were digested with trypsin in-solution followed by LC–MS/MS, allowing a liquid handler-assisted high throughput workflow. In the pretreatment of human serum, one-step purification of glycoproteins with minimal co-isolation of abundant serum proteins including albumin was achieved by using Dynabeads. The novel platform also held the potential to allow transfer to an automated liquid handle.




9

Fig. 4. Illustration of the most commonly used strategies for glycoproteins/glycopeptides enrichment, including lectin-based affinity chromatography, hydrazine chemical reaction, boronate affinity chromatography and hydrophilic interaction liquid chromatography [9].

As an improvement of this study, a semi-automated highthroughput glycoprotein biomarker discovery platform termed lectin magnetic bead array (designated as LEMBA)-coupled MS/MS was developed [103], which included (i) effective single-step serum glycoprotein isolation using a panel of 20 individual lectin-coated magnetic beads in microplate format, (ii) on-bead trypsin digestion, and (iii) nano LC–MS/MS with lectin exclusion list. With optimized steps in the high-throughput workflow to incorporate on-bead tryptic digest and a lectin-exclusion list, processing of 4 samples through a 20 lectin-LeMBA required only 3 h. 4.2. Hydrazine chemical method Different from lectin-based affinity chromatography, hydrazine chemical method can isolate all glycoproteins, regardless of glycan structure [9]. Hydrazine chemical method involved the oxidation of glycans’ cis-diol groups to aldehydes and the covalent coupling between aldehydes and hydrazide-immobilized materials. Nonspherical silica particles containing superparamagnetic Fe3 O4 cores were produced by using a modified Stober method and surface modification of the resulting Fe3 O4 @nSiO2 with hydrazide-terminated silane [104]. The density of hydrazide groups reached 8 nmol/mg while the coupling capacity of glycoproteins reached 36 mg/g, which was five times higher than that of conventional polymer beads. These hydrazide-functionalized Fe3 O4 @nSiO2 microspheres have also been applied to parallel, high-throughput isolation of N-linked glycopeptides from human plasma by being integrated with a 96-well plate and a Tecan liquid handler. This study provoked more research interest in the synthesis of magnetic materials based on hydrazine chemical method. Four approaches have been proposed and compared for the production of hydrazine-functionalized magnetic microspheres by hydrazine

modification on the surface of the carboxyl and epoxy-silanized magnetic particles, respectively [105]. The four core–shell structured microspheres were carboxyl-silanized Fe3 O4 functionalized with hydrazine hydrate or adipic dihydrazide, and epoxy-silanized Fe3 O4 functionalized with hydrate or adipic dihydrazide. The results revealed that adipic dihydrazide functionalized carboxylsilanized magnetic particles displayed the maximum capture selectivity and capability for glycoproteins. The capacity of the composites (1 g) for coupling bovine fetuin was 130 ± 5.3 mg. The capability of this method was also confirmed by successful isolation of all formerly glycosylated peptides from standard glycoproteins and identification of their glycosylation sites. To solve the problem of hydrophilicity and biocompatibility, hydrazide-functionalized poly(2-hydroxyethyl methacrylate) (PHEMA)-modified magnetic microspheres were employed for the specific enrichment of glycoproteins [106]. Due to the bio-friendly properties of PHEMA, the magnetic polymer prepared could effectively reduce non-specific adsorption of non-glycosylated peptides. Especially, the density of the hydrazide groups was as high as 18 ␮mol/mg. The microspheres were also compared with commercial SiMAG-hydrazide particles for capturing the glycoproteins of bacterial origin, such as F. tularensis glycoproteins. Consequently, 50% of all identified F. tularensis putative glycoproteins had already been identified in a previous study [107]. To sum up, hydrazide-functionalized magnetic microspheres offer two advantages for glycoprotein isolation compared with lectin affinity chromatography. Firstly, detergents solubilizing strongly hydrophobic membrane proteins present in the cell lysates can be securely used. Secondly, harsher washing solvents such as organic and chaotropic agents with high concentrations can be applied prior to proteolytic digestion of trapped glycoproteins in order to minimize co-elution of nonspecifically adsorbed nonglycoproteins.




4.3. Boronate affinity chromatography Reversible reactions between boronate and cis-diol-containing structures are present in most sugar moieties. Boronate affinity chromatography based on these reactions is another kind of method extensively utilized to enrich glycoproteins/glycopeptides. The boronic acids are readily available, stable, and versatile, and provide wider sugar binding coverage than traditional lectins [108]. As the first example of boronic acid-functionalized magnetic material for phosphoproteins/phosphopeptides enrichment, aminophenylboronic acid (APBA) was immobilized on the surface of carboxylic acid-terminated magnetic microspheres by coupling with carbodiimide and subsequently reacted with Nhydroxysuccinimide moieties [109]. Glycoproteins, such as HbA1c, fibrinogen, or RNase B were separated and desalted by using APBAfunctionalized magnetic beads. Peptide peaks obtained from RNase B and HbA1c covered 66.7% of amino groups for RNnase B and 82.5% for HbA1c ␤ chains. A facile method to prepare APBA-functionalized Fe3 O4 microspheres were developed by adopting chemical coprecipitation and a multi-step covalent modification [110]. Adsorption experiments and SDS-PAGE demonstrated that the APBA-coated Fe3 O4 microspheres had specific recognition and fast kinetics for glycoproteins. In addition, the practicability of the as-prepared nanocomposites was assessed by specific capture of ovalbumin from an egg white sample. Since multistep surface modifications were rather tedious and time-consuming, APBA-functionalized core–shell structured magnetic microspheres were prepared via a one-pot approach [111], in which tetramethyloxysilane (TMOS) and 3-methacryloxypropyltrimethoxysilane (3-MAPS) served as co-precursors, 4-vinylphenylboronic acid (VPBA) served as functionalized monomer, and ethylene glycol dimethacrylate (EDMA) served as crosslinker. Another simple synthetic route for APBA-functionalized magnetic microspheres was accomplished by the modification of amine-magnetite nanoparticles with hexanedioyl chloride and 3-aminophenylboronic acid [112]. An enrichment factor of 6 was achieved for the mixture of peptides. Mixtures of glycosylated horseradish peroxidase (HRP)/RNase B and nonglycosylated BSA/MYO were used to assess the specificity for glycoproteins enrichment. A recovery of 77.78% was achieved for HRP, and a recovery of 58.39% was achieved for RNase B by the application of APBA-functionalized magnetic microspheres. The amount of boronic acid on magnetic particles has a great influence on the enrichment efficiency for glycoproteins or glycopeptides. Gold nanoparticles were reported to easily immobilize a large amount of 4-mercaptophenylboronic acid due to the robust interaction between the thiol groups and gold nanoparticles [113,114]. Another brilliant feature of gold nanoparticles-incorporated materials is large specific area. With increased surface area, they can capture target molecules to the largest extent. With the aim to improve the amount of boronic acid immobilized on Fe3 O4 particles, Fe3 O4 @CP@Au microspheres were synthesized via a layer-by-layer self-assembly process, followed by the modification of 4-Mercaptophenylboronic acid (MPBA) on the surface [115]. Fe3 O4 @CP@Au magnetic microspheres were successfully applied to the selective enrichment of glycoproteins and glycopeptides. Almost all glycopeptides of HRP with a concentration of 2.5 ng/␮L could be detected after enrichment with the MPBA-immobilized Fe3 O4 @CP@Au microspheres. Several glycopeptides can be detected even when the concentration ratio of HRP and ␤-casein has reached 1:10, exhibiting superior sensitivity and selectivity than previous reports based on boronic acid affinity probes [112,114]. APBA-functionalized Fe3 O4 /Au composites were prepared in the following way. Firstly, Fe3 O4 @nSiO2 cores and gold subcores were

conjugated through 3-mercaptopropylmethyldimethoxysilane (MPMDMS) [116]. Then, the Fe3 O4 @CP@Au particles were redispersed in 11-mercaptoundecanol (MUD) to form a self-assembled monolayer of MUD outside the gold particles. Finally, the hydroxyl groups of MUD were converted into carboxyl groups to provide reaction sites for the grafting of APBA. The adsorption capacity of the composites was calculated to be over 79 mg of glycoproteins per gram, which was 3 times higher than that of the commercial magnetic beads. By using this strategy, the recovery of glycopeptides and glycoproteins after enrichment were found to be 85.9 and 71.6%, separately. In the enrichment of glycopeptides from human colorectal cancer tissues, 194 unique glycosylation sites mapped to 155 different glycoproteins had been identified, of which 165 sites (85.1%) were newly identified. A ligand-free strategy for ultrafast and highly selective enrichment of glycopeptides by using Ag-coated magnetic nanoarchitectures has been developed [117]. The composites were deliberately designed to be constructed with a magnetic colloid nanocrystal cluster (MCNC) core, a poly(methacrylic acid) (PMAA) intermediate layer and a Ag nanoparticle shell with high coverage. Glycopeptides even at a low molar ratio of glycopeptides/nonglycopeptides (1:100) could be identified in 1 min after being enriched by MCNC@PMAA@Ag composites. And 127 unique glycopeptides assigned to 51 different glycoproteins were identified from only 1 mL of rat serum. 4.4. Hydrophilic interaction liquid chromatography (HILIC) HILIC is a promising approach to enrich glycopeptides, due to their relatively higher hydrophilicity toward glycosylated peptides than non-glycosylated peptides. HILIC-based methods have the merits of high selectivity, good reproducibility and non-irreversible alterations of the glycan composition for the enrichment of glycopeptides [9]. However, magnetic microspheres-based HILIC for glycopeptide enrichment had not attracted extensive attention until recently. A rapid and simple method for purification of glycan-binding proteins from human serum was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate [118]. Firstly, the epoxy-coated Fe3 O4 microspheres were hydroxyl functionalized with 4-hydroxybenzhydrazide. Then, carbohydrates were efficiently immobilized on hydroxyl-functionalized magnetic particles by formation of glycosidic bond with the hemiacetal groups at the reducing end of the suitable carbohydrates via condensation. The result showed that the amount coupled with mannose for 1 mg of magnetic particles was 10 mol in acetate buffer (pH = 5.4). The methodology was potential to work together with the glycan microarrays for screening and purification of important GBPs from complex protein samples. Amine-functionalized magnetic microspheres were also prepared for the enrichment of glycopeptides [119]. In this work, the adsorbed glycopeptides could be deglycosylated by peptide N-glycosidase F (PNGase F), which allowed the determination of N-glycosylation sites. The applicability for glycopeptides enrichment was further evaluated with mouse uterine luminal fluid. Forty-three per cent of 209 unique peptides were identified as de-N-glycosylated peptides carrying the characteristic Asn to Asp conversion. Maltose-functionalized magnetic hybrid materials were synthesized by surface-initiated atom transfer radical polymerization (SI-ATRP) of poly(ethylene glycol) (PEG) brushes on the surface of Fe3 O4 @nSiO2 microspheres and subsequent functionalization with hydrophilic maltose groups [120]. The PEG brushes not only enhanced the water solubility of magnetic particles, but also led to three-dimensional scaffolds for the immobilization of hydrophilic maltose, resulting in an enhanced multivalent




HILIC interaction between glycopeptides and the as-prepared Fe3 O4 @nSiO2 @PEG-maltose. A LOD of 0.5 fmol was obtained for tryptic digest of human IgG, which was lower than that treated with boronic acid and hydrophilic silica-based Click maltose (50 fmol) [113,121–123]. Compared with glycoproteins identification without pre-separation by SDS-PAGE, 20 more N-linked glycoproteins were identified by the present method (204 N-glycosylation sites in 106 glycoproteins in all). Zwitterionic (ZIC)-based HILIC was shown to have better hydrophilic interactions toward glycopeptides than other HILIC adsorbents [124,125]. A novel magnetic microspheres-based zwitterionic HILIC (ZIC-HILIC) material was fabricated by coating a zwitterionic polymer synthesized by spontaneous acid-catalyzed polymerization of 4-vinyl-pyridinium ethanesulfonate monomer on polyacrylic acid-coated Fe3 O4 [126]. The resulting magnetic ZICHILIC microspheres provided high specificity (6 pmol of BSA tryptic digest was spiked into 1 pmol of fetuin digest) and high recovery yield (95–100%) for the enrichment of glycopeptides from fetuin. A two-step HILIC enrichment strategy was employed for large scale analysis of glycoproteins in complex biological samples. Eighty-five N-glycosylation sites in 53 glycoproteins from urine samples were identified after enrichment with the magnetic ZIC-HILIC microspheres. Two novel glycosylation sites on N513 of uromodulin and N470 of lysosomal alpha-glucosidase which had not been reported were identified by a two-step HILIC approach. Furthermore, all these identified sites were confirmed by studies using PNGase F deglycosylation and 18 O enzymatic labeling. In conclusion, among functionalized magnetic microspheresbased approaches to the selective enrichment of glycoproteins and glycopeptides, lectin-based materials have unique affinity toward specific glycan moieties, and were successfully applied to the treatment of cell lysate of human hepatocellular carcinoma cell line 7703 and living HepG-2 cells. Hydrazide-functionalized materials can isolate all glycoproteins regardless of glycan structure, and successfully isolated and identified glycopeptides and glycoproteins in human plasma and F. tularensis. Boronic acid-functionalized magnetic materials also provide wider sugar binding coverage, and were employed in the enrichment and identification of egg white, human serum, human colorectal cancer tissues and rat serum. Last but not least, HILIC-based materials possess the merit of nonirreversible alterations of the glycan composition, and were utilized in the pretreatment of mouse uterine luminal fluid, tryptic digest of human IgG and human urine. 5. Conclusions and perspectives Although much progress has been made in the application of core–shell structured magnetic microspheres to the enrichment of low-abundance peptides, the design of novel functionalized magnetic nanocomposites with well-defined nanostructures and surface properties for the application in proteomics remains an area of intense research interest. The feasibility of enrichment must be further verified with more and more real samples where the complexity levels touch higher magnitudes. Acknowledgements This work was supported by the National Basic Research Priorities Program (2012CB910602, 2013CB911201), the National Natural Science Foundation of China (21075022, 21275033, 21105016), Research Fund for the Doctoral Program of Higher Education of China (20110071110007, 20100071120053), the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (J1103304) and Graduate Innovation Fund of Fudan University.

11

References [1] M. Mann, P. Hojrup, P. Roepstorff, Biol. Mass Spectrom. 22 (1993) 338. [2] J.R. Yates, S. Speicher, P.R. Griffin, T. Hunkapiller, Anal. Biochem. 214 (1993) 397. [3] S. Fields, Science 291 (2001) 1221. [4] A. Abbott, Nature 409 (2001) 747. [5] X. Jiang, M. Ye, H. Zou, Proteomics 8 (2008) 686. [6] M. Mann, O.N. Jensen, Nat. Biotechnol. 21 (2003) 255. [7] J.C. Paulson, O. Blixt, B.E. Collins, Nat. Chem. Biol. 2 (2006) 238. [8] T.E. Thingholm, O.N. Jensen, M.R. Larsen, Proteomics 9 (2009) 1451. [9] X. Li, G. Zhu, Y. Luo, B. Yuan, Y. Feng, TrAC – Trends Anal. Chem. 45 (2013) 233. [10] J. Lu, M. Wang, Y. Li, C. Deng, Nanoscale 4 (2012) 1577. [11] Q. Liu, J. Shi, M. Cheng, G. Li, D. Cao, G. Jiang, Chem. Commun. 48 (2012) 1874. [12] Y. Luo, Z. Shi, Q. Gao, Y. Feng, J. Chromatogr. A 1218 (2011) 1353. [13] E.P. Diamandis, J. Proteome Res. 5 (2006) 2079. [14] E.F. Petricoin, C. Belluco, R.P. Araujo, L.A. Liotta, Nat. Rev. Cancer 6 (2006) 961. [15] H. Chen, X. Xu, N. Yao, C. Deng, P. Yang, X. Zhang, Proteomics 8 (2008) 2778. [16] N. Yao, H. Chen, H. Lin, C. Deng, X. Zhang, J. Chromatogr. A 1185 (2008) 93. [17] H. Chen, C. Deng, Y. Li, Y. Dai, P. Yang, X. Zhang, Adv. Mater. 21 (2009) 2200. [18] L. Sun, Q. Zhao, G. Zhu, Y. Zhou, T. Wang, Y. Shan, K. Yang, Z. Liang, L. Zhang, Y. Zhang, Rapid Commun. Mass Spectrom. 25 (2011) 1257. [19] S. Liu, Y. Li, C. Deng, Y. Mao, X. Zhang, P. Yang, Proteomics 11 (2011) 4503. [20] H. Chen, D. Qi, C. Deng, P. Yang, X. Zhang, Proteomics 9 (2009) 380. [21] H. Unal, J.H. Niazi, J. Mater. Chem. B 1 (2013) 1894. [22] G. Cheng, Y. Liu, Z. Wang, J. Zhang, D. Sun, J. Ni, J. Mater. Chem. 41 (2012) 21998. [23] H. Chen, S. Liu, Y. Li, C. Deng, X. Zhang, P. Yang, Proteomics 11 (2011) 890. [24] W. Jia, X. Chen, H. Lu, P. Yang, Angew. Chem. Int. Ed. Engl. 45 (2006) 3345. [25] H. Chen, C. Deng, X. Zhang, Angew. Chem. Int. Ed. 49 (2010) 607. [26] R. Tian, H. Zhang, M. Ye, X. Jiang, L. Hu, X. Li, X. Bao, H. Zou, Angew. Chem. Int. Ed. 46 (2007) 962. [27] R. Tian, L. Ren, H. Ma, X. Li, L. Hu, M. Ye, R. Wu, Z. Tian, Z. Liu, H. Zou, J. Chromatogr. A 1216 (2009) 1270. [28] H. Chen, S. Liu, H. Yang, Y. Mao, C. Deng, X. Zhang, P. Yang, Proteomics 10 (2010) 930. [29] G. Zhu, X. Li, Q. Gao, N. Zhao, B. Yuan, Y. Feng, J. Chromatogr. A 1224 (2012) 11. [30] S. Liu, H. Chen, X. Lu, C. Deng, X. Zhang, P. Yang, Angew. Chem. Int. Ed. 49 (2010) 7557. [31] M. Zhao, C. Deng, X. Zhang, P. Yang, Proteomics 13 (2013) 3387. [32] L. Zhao, Y. Zhang, J. Wei, D. Cao, K. Liu, X. Qian, Proteomics 9 (2009) 4416. [33] Y. Yu, M. Liu, G. Yan, Y. He, C. Xu, H. Shen, P. Yang, Talanta 85 (2011) 1001. [34] X. Zhang, S. Zhu, C. Deng, X. Zhang, Talanta 88 (2012) 295. [35] T. Hunter, Cell 100 (2000) 113. [36] T. Pawson, J.D. Scott, Trends Biochem. Sci. 30 (2005) 286. [37] S. Zolnierowicz, M. Bollen, EMBO J. 19 (2000) 483. [38] M. Carrascal, D. Ovelleiro, V. Casas, M. Gay, J. Abian, J. Proteome Res. 7 (2008) 5167. [39] P. Cohen, Trends Biochem. Sci. 25 (2000) 596. [40] M. Mann, S.E. Ong, M. Gronborg, H. Steen, O.N. Jensen, A. Pandey, Trends Biotechnol. 20 (2002) 261. [41] T. Pawson, J.D. Scott, Science 278 (1997) 2075. [42] M.B. Yaffe, A.E. Elia, Curr. Opin. Cell Biol. 13 (2001) 131. [43] A. Gruhler, J.V. Olsen, S. Mohammed, P. Mortensen, N.J. Faergeman, M. Mann, O.N. Jensen, Mol. Cell. Proteomics 4 (2005) 310. [44] S. Feng, M. Ye, H. Zhou, X. Jiang, X. Jiang, H. Zou, B. Gong, Mol. Cell. Proteomics 6 (2007) 1656. [45] T.E. Thingholm, T.J.D. Jorgensen, O.N. Jensen, M.R. Larsen, Nat. Protoc. 1 (2006) 1929. [46] C.A. Nelson, J.R. Szczech, Q. Xu, M.J. Lawrence, S. Jin, Y. Ge, Chem. Commun. 43 (2009) 6607. [47] J. Lu, C. Deng, X. Zhang, P. Yang, ACS Appl. Mater. Interfaces 5 (2013) 7330. [48] M. Gronborg, T.Z. Kristiansen, A. Stensballe, J.S. Andersen, O. Ohara, M. Mann, O.N. Jensen, A. Pandey, Mol. Cell. Proteomics 1 (2002) 517. [49] B.A. Ballif, J. Villen, S.A. Beausoleil, D. Schwartz, S.P. Gygi, Mol. Cell. Proteomics 3 (2004) 1093. [50] X. Xu, C. Deng, M. Gao, W. Yu, P. Yang, X. Zhang, Adv. Mater. 18 (2006) 3289. [51] Y. Li, D. Qi, C. Deng, P. Yang, X. Zhang, J. Proteome Res. 7 (2008) 1767. [52] S.B. Ficarro, G. Adelmant, M.N. Tomar, Y. Zhang, V. Cheng, J.A. Marto, Anal. Chem. 81 (2009) 4566. [53] Y. Li, Y. Lin, P. Tsai, C. Chen, W. Chen, Y. Chen, Anal. Chem. 79 (2007) 7519. [54] W. Chen, Y. Chen, Anal. Chem. 79 (2007) 2394. [55] C. Lo, W. Chen, C. Chen, Y. Chen, J. Proteome Res. 6 (2007) 887. [56] L. Novotna, T. Emmerova, D. Horak, Z. Kucerova, M. Ticha, J. Chromatogr. A 1217 (2010) 8032. [57] Y. Yan, Z. Zheng, C. Deng, X. Zhang, P. Yang, Chem. Commun. 49 (2013) 5055. [58] H. Zhou, S. Xu, M. Ye, S. Feng, C. Pan, X. Jiang, X. Li, G. Han, Y. Fu, H. Zou, J. Proteome Res. 5 (2006) 2431. [59] J. Wei, Y. Zhang, J. Wang, F. Tan, J. Liu, Y. Cai, X. Qian, Rapid Commun. Mass Spectrom. 22 (2008) 1069. [60] L. Zhao, R. Wu, G. Han, H. Zhou, L. Ren, R. Tian, H. Zou, J. Am. Soc. Mass Spectrom. 19 (2008) 1176.




[61] D. Qi, Y. Mao, J. Lu, C. Deng, X. Zhang, J. Chromatogr. A 1217 (2010) 2606. [62] X. Li, J. Wu, Y. Zhao, W. Zhang, Q. Gao, L. Guo, B. Yuan, Y. Feng, J. Chromatogr. A 1218 (2011) 3845. [63] J. Lu, Y. Li, C. Deng, Nanoscale 3 (2011) 1225. [64] L. Zhang, Q. Zhao, Z. Liang, K. Yang, L. Sun, L. Zhang, Y. Zhang, Chem. Commun. 48 (2012) 6274. [65] X. Li, L. Xu, G. Zhu, B. Yuan, Y. Feng, Analyst 137 (2012) 959. [66] C. Chen, Y. Chen, Anal. Chem. 77 (2005) 5912. [67] F. Tan, Y. Zhang, J. Wang, J. Wei, P. Qin, Y. Cai, X. Qian, Rapid Commun. Mass Spectrom. 21 (2007) 2407. [68] Y. Li, X. Xu, D. Qi, C. Deng, P. Yang, X. Zhang, J. Proteome Res. 7 (2008) 2526. [69] Y. Li, J. Wu, D. Qi, X. Xu, C. Deng, P. Yang, X. Zhang, Chem. Commun. (2008) 564. [70] J. Wu, X. Li, Y. Zhao, Q. Gao, L. Guo, Y. Feng, Chem. Commun. 46 (2010) 9031. [71] S. Feng, Z. Ren, Y. Wei, B. Jiang, Y. Liu, L. Zhang, W. Zhang, H. Fu, Chem. Commun. 46 (2010) 6276. [72] Q. Min, X. Zhang, H. Zhang, F. Zhou, J. Zhu, Chem. Commun. 47 (2011) 11709. [73] Y. Zeng, H. Chen, K. Shiau, S. Hung, Y. Wang, C. Wu, Proteomics 12 (2012) 380. [74] W. Ma, Y. Zhang, L. Li, L. You, P. Zhang, Y. Zhang, J. Li, M. Yu, J. Guo, H. Lu, ACS Nano 6 (2012) 3179. [75] L. Ji, J. Wu, Q. Luo, X. Li, W. Zheng, G. Zhai, F. Wang, S. Lv, Y. Feng, J. Liu, S. Xiong, Anal. Chem. 84 (2012) 2284. [76] H. Li, X. Shi, L. Qiao, X. Lu, G. Xu, J. Chromatogr. A 1275 (2013) 9. [77] Y. Li, T. Leng, H. Lin, C. Deng, X. Xu, N. Yao, P. Yang, X. Zhang, J. Proteome Res. 6 (2007) 4498. [78] Y. Li, Y. Liu, J. Tang, H. Lin, N. Yao, X. Shen, C. Deng, P. Yang, X. Zhang, J. Chromatogr. A 1172 (2007) 57. [79] C. Chen, Y. Chen, Chem. Commun. 46 (2010) 5674. [80] Y. Li, H. Lin, C. Deng, P. Yang, X. Zhang, Proteomics 8 (2008) 238. [81] D. Qi, J. Lu, C. Deng, X. Zhang, J. Phys. Chem. C 113 (2009) 15854. [82] W. Chen, Y. Chen, Anal. Bioanal. Chem. 398 (2010) 2049. [83] H. Lin, W. Chen, Y. Chen, Anal. Bioanal. Chem. 394 (2009) 2129. [84] D. Qi, J. Lu, C. Deng, X. Zhang, J. Chromatogr. A 1216 (2009) 5533. [85] H. Lin, W. Chen, Y. Chen, J. Biomed. Nanotechnol. 5 (2009) 215. [86] G. Cheng, J. Zhang, Y. Liu, D. Sun, J. Ni, Chem. Commun. 47 (2011) 5732. [87] G. Cheng, J. Zhang, Y. Liu, D. Sun, J. Ni, Chem. Commun. 47 (2011) 12889. [88] A. Lee, H.J. Yang, E.S. Lim, J. Kim, Y. Kim, Rapid Commun. Mass Spectrom. 22 (2008) 2561. [89] S. Chen, Y. Juang, M. Chien, K. Li, C. Yu, C. Lai, Analyst 136 (2011) 4454. [90] K. Imami, N. Sugiyama, Y. Kyono, M. Tomita, Y. Ishirama, Anal. Sci. 1 (2008) 161. [91] H.K. Kweon, K. Hakansson, Anal. Chem. 78 (2006) 174. [92] G. Han, M. Ye, H. Zou, Analyst 133 (2008) 1128. [93] M.R. Condina, M.A. Guthridge, S.R. McColl, P. Hoffmann, Proteomics 9 (2009) 3047. [94] C. Chen, L. Wang, Y. Ho, Anal. Bioanal. Chem. 399 (2011) 2795. [95] A.M. Wuhrer, M.I. Catalina, A.M. Deelder, C.H. Hokke, J. Chromatogr. B 849 (2007) 115. [96] L. Zhang, H. Lu, P. Yang, Anal. Bioanal. Chem. 396 (2009) 199.

[97] [98] [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126]

R.D. Cummings, S. Kornfeld, J. Biol. Chem. 257 (1982) 11235. A. Kamra, M.N. Gupta, Anal. Biochem. 164 (1987) 405. K. Sparbier, T. Wenzel, M. Kostrzewa, J. Chromatogr. B 840 (2006) 29. J. Tang, Y. Liu, P. Yin, G. Yao, G. Yan, C. Deng, X. Zhang, Proteomics 10 (2010) 2000. G. Yang, T. Cui, Q. Chen, T. Ma, Z. Li, Anal. Biochem. 421 (2012) 339. D. Loo, A. Jones, M.M. Hill, J. Proteome Res. 9 (2010) 5496. E. Choi, D. Loo, J.W. Dennis, C.A. O’Leary, M.M. Hill, Electrophoresis 32 (2011) 3564. Z. Zou, M. Ibisate, Y. Zhou, R. Aebersold, Y. Xia, H. Zhang, Anal. Chem. 80 (2008) 1228. Z. Li, S.S. Sun, G.L. Yang, T. Wang, Q.Z. Wang, C. Chen, Anal. Bioanal. Chem. 396 (2010) 3071. D. Horak, L. Balonova, B.F. Mann, Z. Plichta, L. Hernychova, M.V. Novotny, et al., Soft Matter 8 (2012) 2775. L. Balonova, L. Hernychova, B.F. Mann, M. Link, Z. Bilkova, M.V. Novotny, J. Stulik, J. Proteome Res. 9 (2010) 1995. Y. Xu, Z. Wu, L. Zhang, H. Lu, P. Yang, P.A. Webley, D. Zhao, Anal. Chem. 81 (2009) 503. J.H. Lee, Y. Kim, M.Y. Ha, E.K. Lee, J. Choo, J. Am. Soc. Mass Spectrom. 16 (2005) 1456. Z. Lin, J. Zheng, F. Lin, L. Zhang, Z. Cai, G. Chen, J. Mater. Chem. 21 (2011) 518. Z. Lin, J. Zheng, Z. Xia, H. Yang, L. Zhang, G. Chen, RSC Adv. 2 (2012) 5062. W. Zhou, N. Yao, G. Yao, C. Deng, X. Zhang, P. Yang, Chem. Commun. (2008) 5577. G. Yao, H. Zhang, C. Deng, H. Lu, X. Zhang, P. Yang, Rapid Commun. Mass Spectrom. 23 (2009) 3493. J. Tang, Y. Liu, D. Qi, G. Yao, C. Deng, X. Zhang, Proteomics 9 (2009) 5046. D. Qi, H. Zhang, J. Tang, C. Deng, X. Zhang, J. Phys. Chem. C 114 (2010) 9221. L. Zhang, Y. Xu, H. Yao, L. Xie, J. Yao, H. Lu, P. Yang, Chem. Eur. J. 15 (2009) 10158. W. Ma, L. Li, Y. Zhang, Q. An, L. You, J. Li, Y. Zhang, S. Xu, M. Yu, J. Guo, H. Lu, C. Wang, J. Mater. Chem. 22 (2012) 23981. X. Sun, G. Yang, S. Sun, R. Quan, W. Dai, B. Li, C. Chen, Z. Li, Curr. Pharm. Biotechnol. 10 (2009) 753. C. Kuo, I. Wu, H. Hsiao, K. Khoo, Anal. Bioanal. Chem. 402 (2012) 2765. Z. Xiong, L. Zhao, F. Wang, J. Zhu, H. Qin, R. Wu, W. Zhang, H. Zou, Chem. Commun. 48 (2012) 8138. L. Yu, X. Li, Z. Guo, X. Zhang, X. Liang, Chem. Eur. J. 15 (2009) 12618. Y. Qu, S. Xia, H. Yuan, Q. Wu, M. Li, L. Zou, L. Zhang, Z. Liang, Y. Zhang, Anal. Chem. 83 (2011) 7457. J. Zhu, F. Wang, R. Chen, K. Cheng, B. Xu, Z. Guo, X. Liang, M. Ye, H. Zou, Anal. Chem. 84 (2012) 5146. K. Neue, M. Mormann, J. Peter-Katalinic, G. Pohlentz, J. Proteome Res. 10 (2011) 2248. L. Mauko, A. Nordborg, J.P. Hutchinson, N.A. Lacher, E.F. Hilder, P.R. Haddad, Anal. Biochem. 408 (2011) 235. C. Yeh, S. Chen, D. Li, H. Lin, H. Huang, C. Chang, J. Chromatogr. A 1224 (2012) 70.


Recent advances in enrichment and separation strategies for mass spectrometry-based phosphoproteomics.

Oxamato-based coordination polymers: recent advances in multifunctional magnetic materials.

Recent Advances in the Application of Magnetic Nanoparticles as a Support for Homogeneous Catalysts.

Recent advances in the mechanical durability of superhydrophobic materials.

Recent advances in topical delivery of proteins and peptides mediated by soft matter nanocarriers.

Recent advances in capillary electrophoresis of proteins, peptides and amino acids.

Interfacial Materials for Organic Solar Cells: Recent Advances and Perspectives.

Recent advances in nanoscale bioinspired materials.

Recent advances in homogeneous and separation-free enzyme immunoassays.

Recent Advances in the Clinical Application of Mass Spectrometry.

Recent advances in the study of zebrafish extracellular matrix proteins.

FLT3 INHIBITORS: RECENT ADVANCES AND PROBLEMS FOR CLINICAL APPLICATION.

Enrichment of phosphorylated peptides and proteins by selective precipitation methods.

Application of Nanostructures in Electrochromic Materials and Devices: Recent Progress.

Recent Advances in Magnetic Microfluidic Biosensors.

Recent Advances in Nanocomposite Materials of Graphene Derivatives with Polysaccharides.

Recent advances in cardiac magnetic resonance.

Recent advances in anisotropic magnetic colloids: realization, assembly and applications.

Interaction of phospholipids with proteins and peptides. New advances 1990.

Recent extensions to native chemical ligation for the chemical synthesis of peptides and proteins.

Recent Advances in the Fabrication and Application of Screen-Printed Electrochemical (Bio)Sensors Based on Carbon Materials for Biomedical, Agri-Food and Environmental Analyses.

Synthesis of polydopamine-coated magnetic graphene for Cu(2+) immobilization and application to the enrichment of low-concentration peptides for mass spectrometry analysis.

Preparation of magnetic hydroxyapatite clusters and their application in the enrichment of phosphopeptides.

Recent advances in pulp capping materials: an overview.