Investigating macromolecular complexes using top-down mass spectrometry.

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DOI 10.1002/pmic.201300333

REVIEW

Investigating macromolecular complexes using top-down mass spectrometry Elisabetta Boeri Erba Institute of Structural Biology (Institut de Biologie Structurale), Centre National de la Recherche Scientifique ´ (CNRS), University of Grenoble Alpes (Universite´ de Grenoble Alpes), Commissariat a` l’Energie Atomique et aux ´ Energies Alternatives (CEA), DSV, Grenoble, France MS has emerged as an important tool to investigate noncovalent interactions between proteins and various ligands (e.g. other proteins, small molecules, or drugs). In particular, ESI under so-called “native conditions” (a.k.a. “native MS”) has considerably expanded the scope of such investigations. For instance, ESI quadrupole time of flight (Q-TOF) instruments have been used to probe the precise stoichiometry of protein assemblies, the interactions between subunits and the position of subunits within the complex (i.e. defining core and peripheral subunits). This review highlights several illustrative native Q-TOF-based investigations and recent seminal contributions of top-down MS (i.e. Fourier transform (FT) MS) to the characterization of noncovalent complexes. Combined top-down and native MS, recently demonstrated in “high-mass modified” orbitrap mass spectrometers, and further improvements needed for the enhanced investigation of biologically significant noncovalent interactions by MS will be discussed.

Received: July 31, 2013 Revised: April 3, 2014 Accepted: April 8, 2014

Keywords: Intact protein assemblies / Native MS / Noncovalent complexes / Technology / Topdown MS

1

Introduction

Living organisms, capable of reproduction and growth, are able to respond to stimuli and to maintain their homoeostasis. This is possible because noncovalent interactions exist between molecules, and many proteins are involved in these cellular communications. Proteins communicate via physical interactions and need to specifically recognize their interacting partners in order to convey their messages. They dock their partners through short-range, noncovalent interactions, such as van der Waals forces, hydrogen bonds, and hydrophobic interactions. Through these interactions, proteins bind ligands such as other proteins, small molecules or drugs, carbohydrates, DNA and RNA. Correspondence: Dr. Elisabetta Boeri Erba, Institute of Structural Biology, University of Grenoble Alpes, F-38000 Grenoble, France 6, rue J. Horowitz, 38000 Grenoble, France E-mail: [email protected] Abbreviations: ADH, alcohol dehydrogenase; AK, adenylate kinase; ATP, adenosine-5’ triphosphate; CAD, collisionally activated dissociation; ECD, electron capture dissociation; ETD, electron transfer dissociation; FT-ICR, Fourier transform ion cyclotron resonance; HCD, higher-energy C-trap dissociation; QTOF, quadrupole time of flight

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Recently, MS has become a valuable approach to investigate noncovalently bound complexes. MS presents several advantages compared to other techniques, particularly in terms of sensitivity and speed of analysis. Using specific conditions to preserve noncovalent interactions [1, 2] (i.e. by native MS), one can determine the mass of intact assemblies, their precise stoichiometry (an aspect inaccessible by other approaches), the direct binding between subunits, the position of subunits within an assembly (i.e. core vs. peripheral subunits) [3–5] and the strength of interactions [6] (Fig. 1). By mixing subunits in a stepwise manner, a hierarchy in the assembly of a complex can be determined [7]. By using appropriate bioinformatics tools, it is even possible to gain insights into the evolution of homo- and hetero-oligomeric protein complexes [8,9]. When native MS is coupled with ion mobility, additional information regarding the shape of noncovalent complexes can be obtained [10]. “Protein footprinting” represents another important MS-based approach to investigate protein structures through chemical modifications [11]. For example, protein conformation and interactions can be assessed using selectively labeling, cleaving residues, or carboxyl group modifications [12]. Colour Online: See the article online to view Fig. 1 in colour.

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Figure 1. A 2D interaction network of subunits within a protein complex can be generated through a multistep process using native MS. Step 1 (composition): under denaturing conditions, the subunits are chromatographically separated and their masses are determined. Step 2 (stoichiometry): using MS conditions optimized for preserving noncovalent interactions (e.g. using ammonium acetate buffer), the measured mass of an intact complex (or a subcomplex) reveals the stoichiometry of the subunits. Step 3 (subunit position): a series of MS/MS spectra indicates whether the subunits are located within the core or at the periphery of the assembly. Step 4 (direct interactions): by adding organic solvent, overlapping subcomplexes (e.g. dimers, trimers) are generated [9, 112]. The composition of the different subcomplexes reveals the direct interactions between the subunits. Step 5 (assembly pathway): individual subunits can be mixed in solution and a mass shift can be detected if a subcomplex is formed. Step 6 (two-dimensional map of interactions): combining all these data allows one to draw an accurate interaction network of a protein complex.

“Top-down” MS represents a key method for the characterization of different proteoforms [13], protein conformations and PTMs. There are several excellent review articles describing recent developments and applications of top-down MS for protein analysis [14–18]. The focus of this review is to illustrate the progress of top-down MS for the study of noncovalent interactions. Quadrupole-time of flight (Q-TOF) spectrometers have been predominately used for investigations of macromolecular assemblies. However, recent developments in “native topdown MS” using FT instruments, allowed for both the analysis of intact assemblies and their fragmentation in the gas phase. Therefore, FTMS technology enables the investigation of quaternary and primary structure within a single instrument. Finally, future perspective on the study of intact macromolecular complexes by MS will be presented.


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Native MS for the study of macromolecular complexes

To carry out native MS analysis of protein assemblies, the sample should be appropriately ionized. With some exceptions [19], mainly nano-electrospray (nano-ESI) (i.e. a flow rate of 20–200 nL/min) [20–24] is used for investigating noncovalent interactions [25,26]. Native MS is typically carried out using volatile buffers such as ammonium acetate [1,27]. Compared to other types of MS analyses, neither acidic conditions nor organic solvents are used. Generally, to study noncovalent interactions, it is necessary not only to use appropriate ionization conditions, but also instruments that are specifically modified to transmit and detect such large assemblies. To date, Q-TOF instruments have been extensively used for the analysis of macromolecular complexes because they display high sensitivity at high m/z (mass over charge


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Proteomics 2014, 14, 1259–1270 Table 1. Comparison of the instruments used to characterize noncovalent complexes

Q-TOF instrument

FT-ICR mass spectrometer

Orbitrap instrument

m/z range Resolution

30 000 5000 FWHM

15 000 High; it depends on the magnet strength

Dissociation Fragmentation

Parent ion selection Very limited

No parent ion selection Highly informative

24 000 25 000 FWHM at m/z 5000 16 000 FWHM at m/z 10 000 Parent ion and monomer selection Highly informative

In this table, the term “dissociation” indicates that noncovalent interactions are broken. This can be used to gain insights into the quaternary structure of a protein (see Fig. 1). “Fragmentation” indicates that covalent bonds are cleaved. This can provide information about the primary structure of a protein (see Figs. 3 and 4). FWHM, full width at half maximum.

ratio). Modifications to the front-end of Q-TOF instruments enabled the detection of protein complexes [28, 29]. In particular, instrumental modifications [e.g. (i) controlling the pressure in the first vacuum stage, (ii) reducing the frequency of quadrupole analyzer, and (iii) relatively high pressure within the collision cell] allowed MS/MS experiments [28–30], whereby intact complexes are dissociated in the gas phase (i.e. noncovalent interactions are broken). Tandem MS is often used to confirm the stoichiometry and provide an insight into the location of the subunits within the complex (core vs. peripheral subunits) [31] (Fig. 1) as the peripheral subunits are expelled from the complex at lower energy compared to the core subunits [7]. Moreover, MS/MS could provide structural information on subunit interactions and relative spatial arrangements [32]. Q-TOF based native MS enabled the investigation of large protein complexes (e.g. an intact 18 MDa viral capsid [33]) and the study of interactions involving membrane proteins (e.g. drug–protein interactions [34], lipid–protein complexes [35], and protein assemblies [36, 37]). Only recently intact heteromeric membrane protein complexes, containing both soluble and transmembrane domains, were analyzed [38–40]. The investigations described above exploited MS/MS and the dissociation of noncovalent interactions to determine the stoichiometry of assembly and interaction networks (Fig. 1 and Table 1). In this review, I will use the term “dissociation” to imply that only noncovalent interactions are broken, whereas “fragmentation” indicates the breakage of covalent bonds. There are examples where Q-TOF instruments induced the fragmentation of constituent subunits [41]. For instance, Pagel et al. investigated the effect of charge state on the collisionally activated dissociation (CAD) of a tetrameric complex, the human transthyretin [42]. Specifically, charge-reduced subunits underwent backbone cleavage on C-termini, prior to dissociation of noncovalent bonds holding the assembly together. On the contrary, highly charged states exhibited the ejection of either unfolded or folded monomers without any evidence of subunit fragmentation. To conclude, “high-mass modified” Q-TOF instruments have been successfully utilized to study soluble and membrane macromolecular complexes and decipher their quaternary structures and interaction networks. Moreover, in a few cases these mass spectrometers were used to characterize the primary structure of proteins within assemblies. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Electron-based fragmentation techniques for top-down MS

“Top-down” MS allows for the characterization of different proteoforms [13], protein conformations, PTMs and protein– ligand binding site locations [14–18]. The term “top-down” MS was coined at the end of the 1990s [43, 44]. An illustrated timeline shows key events in connection with top-down MS (Fig. 2). The development of ESI [45], of nano-ESI [46] and the ESI-FT-ICR analysis of intact proteins [47] laid the foundations for top-down MS. This type of MS originated from the development of electron capture dissociation (ECD) in 1998 [43] and then intact proteins were studied by top-down MS [44]. Afterwards, small noncovalent complexes [48], a ligand– protein complex [49] and subunits still bound to an intact protein assembly [50] were fragmented by ECD (see below). Recently, highly charged monomers, ejected from intact protein complexes, were broken down into pieces by performing MS3 experiments [51] (see below). Fragmentation in “top-down” MS is accomplished by ECD and electron transfer dissociation (ETD). ECD induces the fragmentation of gas-phase ions because trapped multiply protonated molecules capture low energy electrons [43, 52]. Due to this capture, odd-electron ions are formed and then fragmented. ECD produces primarily c- and z-type fragment ions and appears to be very bond specific. It was observed that peptide backbone cleavages take place and weak interactions (e.g. involving PTMs or noncovalent ligand bindings) can be preserved [53]. To date, ECD has been mainly used in FTICR MS, but has also been incorporated in ion trap [54] and Q-TOF spectrometers [55]. Similar to ECD, ETD is a fragmentation method based on capture of electrons originating from radical anions [56]. ETD induces the peptide backbone fragmentation generating c- and z-type ions, and preserves labile PTMs [57]. Using ETD, Tsybin and colleagues analyzed 150 kDa monoclonal antibodies (immunoglobulins G, IgGs) under denaturing conditions, fragmenting macromolecules much larger than typically achieved by ordinary ETD/ECD [58]. In these experiments, high-resolution spectra enabled good sequence coverage, revealing features of the IgG variable domains. Recent work carried out by Tsybin’s group, employing top-down LC-ETD on an orbitrap [59], and by Marshall’s group, using ECD on an FTICR [60], suggest that the sequence coverage www.proteomics-journal.com

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Figure 2. Timeline of top-down MS. The advent of ESI [45], of nano-ESI [46] and the ESI-FT-ICR analysis of intact proteins [47] laid the groundwork for top-down MS. In 1998 electron capture dissociation (ECD) represented a key development in the field [43]. Then, proteins were studied by top-down MS [44] and a software specific for top-down MS was developed [113]. In 2002 noncovalent complexes were fragmented by ECD [48]. Then electron transfer dissociation (ETD) was developed [56]. In 2006 ECD fragmentation of a ligand–protein complex [49] and top-down analysis of proteins larger than 200 kDa [114] were carried out. Afterwards, intact protein complexes were fragmented by ECD [50] and in 2013 subunits ejected from intact protein complexes were broken down using MS3 experiments [51].

obtained by ETD and ECD are comparable [60]. Overall, ECD and ETD represent diagnostic fragmentation approaches that complement the most widely used CAD.

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Native top-down MS investigation of noncovalent complexes containing small ligands

Electron-based fragmentation [52] (i.e. ECD and ETD) allows the preservation of noncovalent bonds, while covalent bonds are fragmented. This remarkable feature enabled the characterization of (i) binding between proteins and different types of ligands (i.e. other proteins and small molecules) and (ii) intramolecular protein interactions. Regarding the study of the protein–ligand bindings, these noncovalent interactions have been analyzed by native ESI coupled with FT-ICR spectrometers. In 2001 Kitova et al. investigated the weak binding of a toxin to mono- and polyvalent oligosaccharides and estimated the association constant for the toxin-ligand complexes [61]. It is worth noting that the high resolution offered by FT-ICR enabled the detection of interactions between a large protein oligomer (i.e. pentamer) and small ligands in a number of copies ranging from one to six. Similar investigation was carried out to study the competitive binding of inhibitors to bovine carbonic anhydrase I1 [62]. In 2002, as one of the first examples of noncovalentbinding maintained intact by ECD, Haselmann et al. investigated a peptidic homo-dimer and the interactions between two glycopeptide antibiotics (i.e. vancomycin and eremomycin) and their target, a bacterial tripeptide (KAA) [48]. This study confirmed that ECD can break strong covalent bonds, while weak bonding within a noncovalent complex is preserved (Fig. 3A). For example, in the case of the KAAvancomycin complex, only 27% of the product ions were due to covalent bond fragmentation. Moreover, this investigation illustrated that different information regarding the gas-phase structure of noncovalent complexes (e.g. position of the bind-


ing site, primary sequence information) can be determined by ECD experiments (Fig. 3). ECD has also been used to investigate the direct interactions of a neuropeptide (i.e. Substance P) with several divalent metals [63]. Remarkably, distinct fragmentation patterns were detected, when different metals were analyzed. For instance, the fragmentation of Zn-containing assemblies determined the formation of product ions due to cleavage of backbone amine bonds (c- and z-type ions). In contrast, when Cu-binding peptides were analyzed, amide bond cleavages determined the formation of b- and y-type ions. Other fragmentation pathways were opened using Co and Ni (see Fig. 4 of [63]). Another example of mapping metal-binding sites on proteins was carried out by Erales et al. [64]. The primary binding sites of transition-metal anticancer drugs (i.e. cisplatin, transplatin, and oxaliplatin) on ubiquitin were also investigated using ECD [65]. Loo and coworkers successfully studied the binding of a noncovalent complex formed by a small disordered protein (␣-synuclein) and a polycationic aliphatic amine (spermine) [49]. Using a 7-Tesla linear ion trap FT-ICR hybrid mass spectrometer, the authors exploited the capability of ECD to characterize the contacts between a protein and its ligands within a noncovalent assembly. Both apoprotein and holo-protein were analyzed. Notably, z- and y-ions, specifically observed for the holo-protein, indicated that the spermine was bound near the C-terminus of the ␣-synuclein. These results were consistent with a previous investigation of the ␣-synuclein– spermine interaction by NMR spectroscopy, indicating that some C-terminal residues were directly involved in the spermine binding. Loo’s group also studied the interactions between a kinase [i.e. the chicken adenylate kinase, AK] and a nucleoside triphosphate (i.e. adenosine-5 triphosphate, ATP) to localize the ATP-binding pocket [66] (Fig. 3B). Using both CAD and ECD, they analyzed the apo and holo forms of the AK. By CAD of the AK-ATP, many distinct b- and y-product ions were generated. For instance, there were product ions retaining intact ATP and a diphosphate group, which allowed the identification of the binding pocket. ECD was coupled with infrared laser heating of the product ions to enhance the


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Figure 3. Fragmentation experiments of noncovalent complexes carried out by Haselmann et al. [48] and by Yin et al. [66]. (A) A peptidic homo-dimer and two peptide-antibiotic complexes were fragmented by ECD. These experiments determined (i) charges reduction, (ii) dissociation of noncovalent bonds, and (iii) fragmentation of covalent bonds. The products resulting from these three events provided important information regarding the gas-phase structure of the complexes (e.g. mass confirmation and primary sequence information). (B) CAD and ECD experiments of an enzyme-ATP complex were carried out to determine the binding site of the nucleoside triphosphate [66].

yield of product ions. The ECD results, combined with the information obtained by CAD, allowed Loo and coworkers to reach a high sequence coverage (61%) of apo-AK. This study epitomizes how top-down MS allows the determination of nucleotide-binding sites of uncharacterized kinases. The combination of CAD and ECD was successful in determining the ligand-binding sites of other noncovalent assemblies. For instance, Zn binding to the 29 kDa carbonic anhydrase and ATP-adenylate kinase complex were investigated [67]. In these analyses, supercharging reagents (e.g. sulfolane and m-nitrobenzyl alcohol) were used to generate an analyte carrying higher number of charges compared to ordinary native MS conditions [67]. The product ion spectra indicated that the dissociation of higher charged complexes yielded more ligand-retaining products (i.e. holo-product ions). Both ECD and CAD were also utilized to characterize nanoscale bilayers (i.e. nanodiscs) formed of lipids (i.e. 1,2-dimyristoyl-snglycero-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine) and of a scaffold protein [68].


A metallodrug-protein complex oxaliplatin-ubiquitin assembly was investigated using a combination of gas-phase dissociative approaches: CAD, ETD, and higher-energy C-trap dissociation (HCD) [69]. Comparison of these approaches revealed that ETD was the most useful for identifying the metal-binding sites. Another interesting example of comparison of fragmentation approaches (ETD vs. ECD) was carried out by Jackson et al. [70]. In particular, the binary interactions between two basic peptides (i.e. YGGFLRR and SFKRRRSSK) and two acidic peptides (i.e. A3 YA3 and GpSSEDLKKEE) were investigated. Both ETD and ECD induced the formation of intramolecular fragments carrying intact electrostatic interactions. The two approaches yielded similar intramolecular fragments in terms of abundance and type [70]. Many noncovalent complexes investigated by ECD were held together by electrostatic binding (e.g. ion pair interactions and salt bridging). However, recently a nonelectrostatic interaction between a protein (i.e. p53-inhibitor protein anterior gradient-2, AGR2) and a peptide (i.e. PTTIYY)


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was studied to characterize the binding site [71]. No permanent electrostatic interactions were involved in this peptide– protein interaction because the hexapeptide was not charged at physiologic pH. Regarding the study of the intramolecular noncovalent interactions, ECD facilitated the characterization of the kinetic intermediates during the folding of proteins in the gas phase [72,73]. Since ECD breaks the protein backbone and preserved the noncovalent bonds, these cleavage events indicated the presence of intramolecular interactions determined by the tertiary structure of a protein. To conclude, ETD and ECD in combination with other fragmentation approaches allowed the detailed characterization of noncovalent complexes, yielding crucial information about the interactions between proteins and small ligands such as metals and nucleotides.

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Figure 4. Fragmentation experiments of a noncovalent protein complex [50]. (A) Full spectrum of the ADH subjected to ECD. The small panel illustrates the spectrum of ADH prior to fragmentation. (B) Zoom of the ECD product-ion spectrum at low m/z range. [These spectra were reproduced with permission from [50] C (2010) Springer]. (C) The ECD experiments on intact protein complexes were carried out with or without parent ion selection. These allowed one to confirm the complex mass and to gain insight into the flexibility of the subunits and into the primary sequence of the proteins forming the assembly.


Native top-down MS investigation of intact protein assemblies

To characterize intact protein complexes, Q-TOF instruments are often used to determine the stoichiometry of protein assemblies and their two dimensional spatial organization. However, it is emerging that large noncovalent assemblies can be analyzed by native ESI coupled with a FT-ICR MS [24]. For instance, this approach was used to follow the pHdependent disassembly of a multimeric metalloenzyme [74]. As the pH was lowered, first all Mg2+ cations were lost from the holo-tetramer, then the apo-tetramer dissociated into trimers, dimers, and monomers. While in this example, no fragmentation was attempted, ECD of large noncovalent assemblies has been demonstrated. For instance, in 2006 Geels et al. investigated a 84-kDa heptameric cochaperonin (i.e. the gp31 complex) using both ECD and sustained off-resonance irradiation CAD [75]. After the gp31 assembly parent ions were selected in the ICR cell employing “stored waveform inverse Fourier transform pulses”, the charge states of parent ions were reduced, due to electron capture without any backbone fragmentation, simply creating a radical complex. In 2010, Gross and coworkers were able to fragment subunits within a protein assembly using ECD [50] for the first time. Specifically, they analyzed the tetrameric yeast alcohol dehydrogenase (ADH) using an ECD-enabled ESI-FT-ICR instrument [50]. They reported the fragmentation of the ADH subunits (i.e. breaking covalent bonds within the monomers) (Fig. 4), thus demonstrating the ability to obtain information regarding both the primary and quaternary structures in the same analysis. Contrary to Geels’ experiments [75], Gross and coworkers subjected all ADH ions to fragmentation, not just a preselected ion population [50]. They also speculated that the lack of ECD fragmentation reported in [75] could be caused by a limited overlap between the preselected ions and the electron beam within the ICR cell [50]. In 2011, the Gross’ group further expanded the number of protein assemblies analyzed www.proteomics-journal.com

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by a FTICR mass spectrometer ranging from 103 kDa (i.e. concanavalin A) to roughly 140 kDa (i.e. Fenna Matthews Olson antenna protein complex) [76]. Zhang et al. used different types of dissociative approaches to extract primary sequence information not only for terminal regions of high flexibility, but also for internal/middle regions. By combining “in-source dissociation” and ECD, the authors monitored the unfolding of ADH subunits [76]. By analyzing different ions, they determined that the gas-phase protein unfolding took place initially at the N-terminus and then moved toward the complex core. Since the ECD-based approach allowed only the identification of the high flexibility terminal regions, Gross and coworkers used CAD to fragment flexible regions in the middle of a protein. Interestingly, the flexibility in the ADH structure, determined by MS, correlated well with the so-called “B-factor,” a parameter obtained by X-ray crystallography and predictive of the flexible regions of a protein. These examples highlight the ability of FT-ICR MS to provide remarkable information not only on interactions between proteins within protein assemblies (Table 1), but also on subunit primary sequence and structural flexibility in the gas phase. In the year 2000 a new type of mass analyzer, called orbitrap, was introduced. It provides resolution and mass measurement accuracy matching that of FT-ICR, without the constraint of a superconducting magnet [77–80]. Principles and applications of orbitrap instruments were reviewed elsewhere [81–83]. In 2012, Heck and coworkers were the first to demonstrate the use of an Orbitrap mass spectrometer (Exactive PlusTM , Thermo Fisher Scientific) for analysis of intact protein complexes ranging from 150 kDa to 800 kDa [84–87] (Fig. 5 and Table 1). To carry out native MS analyses, a commercial instrument was modified as follows [84]. (i) The mass range was extended to m/z 24 000 by modifying the control software to increase voltages applied to the RF multipoles, including the C-trap. (ii) The HCD cell was used to efficiently desolvate and trap large macromolecules. (iii) The pressure in HCD cell was manually regulated to allow the use of two collision gases (either nitrogen or xenon) and the control of the pressure in the Orbitrap, which ranged between 5 × 10−10 and 2 × 10−9 mBar. (iv) Voltages of the RF multipoles and ion lenses were manually regulated to select the mass(es) of the macromolecular ions. This modified mass spectrometer reached a very high sensitivity, enabling the analysis of IgG at 1 nM concentration. It also presented a remarkable resolution at high m/z (i.e. full width at half height of 16 000 at m/z = 10 000) allowing for the characterization of different glycosylation forms of IgG [85]. Similarly, due to high resolution, it was also possible to determine the type (i.e. adenosine 5 -diphosphate or ATP) and the number of nucleosides when these were bound to a large assembly (i.e. GroEL, 800 kDa). In 2013, Kelleher’s group further modified an Exactive Orbitrap mass spectrometer [51] to perform MS3 experiments on intact protein complexes [51]. This means that protein assemblies were dissociated (i.e. in MS2 experiments) and then the ejected monomers were further fragmented in the HCD C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 5. Mass spectra of intact macromolecules acquired using a modified Orbitrap. Spectra of (A) an IgG antibody [a zoom of 25+ charge state of IgG1 is also shown]; (B) oligomers of bacteriophage HK97 capsid; (C) yeast 20S proteasome (D), and E. coli GroEL. Crystal structures are also presented. [ReproC (2012) Nature Publishing duced with permission from [84] Group].

cell (in MS3 experiments). Finally, the fragment ions were transferred into the C-trap and Orbitrap to record spectra at high resolution and accuracy. The MS2 experiments allowed the confirmation of the protein complex stoichiometry and the MS3 enabled the characterization of primary sequence of the ejected subunit. In summary, modifications of hardware and pressure of ESI-Q-orbitrap instruments allowed the investigation of primary and quaternary protein structures, using a single instrument. It is reasonable to expect that similarly powerful mass spectrometers, specifically modified for high masses analysis, will become commercially available in the near future.

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Perspective and concluding remarks

MS has greatly contributed to our current knowledge in biology. Today it is possible to quantify the majority of the proteins present in certain organisms [88] or on their cellular surface [89] using MS-based proteomics [90]. Using cross-linking technologies, it is feasible to investigate protein interactions and topologies not only in vitro but also in cells [91]. Despite these impressive technological advances, www.proteomics-journal.com

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many challenges remain. For instance, the extent and nature of protein variations could remain elusive when using “bottom-up approaches.” These large-scale proteomic studies should be complemented by “top-down” MS investigations to better characterize different proteoforms [13]. Moreover, it has become progressively clear that a cell population is quite heterogeneous and population-averaged data can be misleading when the goal is to characterize molecular mechanisms. Therefore, in the long term, we should aim at highthroughput single cell MS to generate detailed views of the cellular responses to specific conditions for large number of individual cells [92, 93]. In addition, determining stoichiometry and interaction networks of protein complexes is crucial for understanding the function of cellular nanomachines. However, to analyze intact macromolecular assemblies directly from individual cells we need better instrumentation in terms of sensitivity, resolution, and ionization efficiency. Orbitrap instruments represent an improvement for their sensitivity and resolution [84,85,87]. However, nano-ESI represents the dominant ionization source for the investigation of intact macromolecular complexes. Novel ways of softly ionizing, desorbing, and nebulizing samples have been developed, eventually increasing the sample fraction introduced in mass spectrometers [94–98]. Similarly, novel types of detector could have a positive impact on native MS. For instance, nanoelectromechanical systems (NEMS) can detect very high masses with unprecedented sensitivity [99]. Only several hundred single-molecule adsorption events are necessary for the analysis of megadalton molecules. Another example is the charge detection mass spectrometry (CDMS) whereby the m/z and z are simultaneously measured for each ion [100]. Furthermore, as a complement to CAD, surface-induced dissociation and ultraviolet photodissociation carried out on large intact proteins and protein complexes look promising to gain structural insights from gas-phase experiments [101–103]. Sample preparation (i.e. the experimental steps that take place before MS analysis) should be improved to increase the yield of macromolecular assemblies purified from cells at their natural level of expression [104, 105]. Another fundamental step in the characterization of macromolecular assemblies is the data analysis and extraction of biological knowledge. While there are several software packages currently available (e.g. [106, 107]), manual evaluation is still unavoidable. To carry out high throughput investigation of macromolecular complexes, we will need software as powerful as tools developed for bottom-up proteomics. During the last decades structural biology has significantly contributed to elucidation of the structure and function of macromolecular assemblies using X-ray crystallography, NMR spectroscopy and electron microscopy [108]. However, these demanding investigations could be greatly facilitated by MS, due its sensitivity and selectivity (i.e. several species with different masses can be simultaneously analyzed). By combining MS with traditional structural biology approaches [109], we will be able to characterize macromolecular com C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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plexes whose structure is unknown and difficult to study using a single approach. An example is the investigation of a polydisperse system formed by ␣B-crystallin [110]. Recently, information obtained by NMR and electron microscopy was combined with MS, ion mobility data to structurally characterize the main oligomeric states of this chaperone [111]. In conclusion, MS is continuously increasing its importance for the characterization of intact noncovalent complexes. Better instruments, improved sample preparation, and automated data interpretation will help MS to become even more significant in our quest to depict the function of nanomolecular machines within living cells. I thank Peter Roepstorff (University of Southern Denmark), Sebastien Hentz (CEA, Grenoble, France), Frank Sobott (University of Antwerpen, Belgium) and Frederic Cazals (INRIA Sophia Antipolis, France) for their critical evaluation of the manuscript. I also acknowledge Joanna Timmins and other members of the Viral Infection and Cancer Group at the IBS for useful discussions. This work was financially supported by the French Infrastructure for Integrated Structural Biology Initiative (FRISBI, ANR-10-INSB-05-02) and by the French National Centre for Scientific Research (CNRS). The author has declared no conflict of interest.

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Characterization of native protein complexes using ultraviolet photodissociation mass spectrometry.

Mass spectrometric analysis of intact macromolecular chaperone complexes.

Investigating the Transformations of Polyoxoanions Using Mass Spectrometry and Molecular Dynamics.

Visualizing the Ensemble Structures of Protein Complexes Using Chemical Cross-Linking Coupled with Mass Spectrometry.

Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry.

Measuring spatial restraints on native protein complexes using isotope-tagged chemical cross-linking and mass spectrometry.

Mass spectrometry of protein complexes: from origins to applications.

Applying mass spectrometry to study non-covalent biomolecule complexes.

Ion spray-tandem mass spectrometry of supramolecular coordination complexes.

Quantifying epitope presentation using mass spectrometry.

Intact glycopeptide characterization using mass spectrometry.

Identification of carotenoids using mass spectrometry.

Detection of gunshot residues using mass spectrometry.

Mass spectrometric methods to analyze the structural organization of macromolecular complexes.

Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling.

Macromolecular ion accelerator mass spectrometer.

Detection of noncovalent FKBP-FK506 and FKBP-Rapamycin complexes by capillary electrophoresis-mass spectrometry and capillary electrophoresis-tandem mass spectrometry.

Rapid high mass resolution mass spectrometry using matrix-assisted ionization.

Investigating the synthesis of ligated metal clusters in solution using a flow reactor and electrospray ionization mass spectrometry.

Panorama of ancient metazoan macromolecular complexes.

Molecular dynamics simulations of large macromolecular complexes.

Soluble macromolecular complexes involving bacterial teichoic acids.

Investigating the formation of "molybdenum blues" with gel electrophoresis and mass spectrometry.

Mass Spectrometry-Based Proteomics for Investigating DNA Damage-Associated Protein Ubiquitylation.