Research article Received: 27 December 2013

Revised: 26 February 2014

Accepted: 5 March 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3357

A method combining SPITC and 18O labeling for simultaneous protein identification and relative quantification Wenlong Zhang,a† Jia Long,a† Cheng Zhang,b Naixuan Cai,a Zhonghua Liu,a Ying Wang,a Xianchun Wang,a Ping Chena* and Songping Lianga* The relative quantification and identification of proteins by matrix-assisted laser desorption ionization time-of-flight MS is very important in /MS is very important in protein research and is usually conducted separately. Chemical N-terminal derivatization with 4-sulphophenyl isothiocyanate facilitates de novo sequencing analysis and accurate protein identification, while 18O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. In the present study, a method combining 4-sulphophenyl isothiocyanate derivatization with 18O isotopic labeling was established to identify and quantify proteins simultaneously in one experiment. Reaction conditions were first optimized using a standard peptide (fibrin peptide) and tryptic peptides from the model protein (bovine serum albumin). Under the optimized conditions, these two independent labeling steps show good compatibility, and the linear relativity of quantification within the ten times dynamic range was stable as revealed by correlation coefficient analysis (R2 value = 0.998); moreover, precursor peaks in MS/MS spectrum could provide accurate quantitative information, which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely, and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. Finally, complex protein mixtures of spider venoms from male and female species with unknown genome information were analyzed. Differentially expressed proteins were successfully identified, and their quantitative information was also accessed. Taken together, this protein identification and quantification method is simple, reliable and efficient, which has a good potential in the exploration of peptides/proteins from species with unknown genome. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: 4-sulphophenyl isothiocyanate; 18O labeling; de novo sequencing; peptide quantification; mass spectrometry

Introduction

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With the development of ‘soft’ ionization methods, including electrospray ionization (ESI) and matrix-assisted laser desorption/ ionization (MALDI), mass spectrometry (MS) provides a particularly sensitive and high-throughput analytical platform for the efficient identification and quantification of expressed proteins.[1–3] The numbers of fully-sequenced genomes are increasing rapidly.[4] However, protein identification by MS without sequence information is difficult, because the probability of unambiguous peptide identification is significantly lower than expected. In earlier studies, MALDI collision-induced dissociation analysis was used for peptide sequencing. However, without any preprocessing, peptide fragmentations (including N-terminal, C-terminal and internal cleavage ions) were random, and the spectrum appeared fairly complex. Subsequently, N-terminal derivatization was applied to reduce the complexity of MS/MS spectra.[5–12] In particular, derivatization with 4-sulphophenyl isothiocyanate (SPITC) generates spectra mainly composed of y-ions and facilitates de novo sequencing analysis, both automatic and manual.[13–16] Previously, a combination of SPITC derivatization and[13] C-labeling was used for identification and relative protein quantification in LC-MS/MS analysis of peptide mixtures.[5] However, the process of synthesis of specific isotope reagents was complicated.

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SPITC modifications were conducted through hours of organic chemical reactions at temperatures as high as 180 °C. Among the various isotopic labeling techniques for relative quantification, 18 O labeling is simple, specific and cost effective for a wide range of analyses. The procedure involves 16O or 18O labeling of a proteolytic digestion mixture, which generates a population of peptide pairs differing in only one or two oxygen atoms at the carboxyl termini.[17–21] Considering the merits of SPITC labeling in sequencing and 18O labeling in quantification, a novel method involving combinative use of SPITC derivatization and 18O labeling was developed. During MALDI analysis, the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) was used as an alternative to α-cyanohydroxycinnamic acid (CCA), because it

* Correspondence to: Ping Chen and Songping Liang, College of Life Sciences, Hunan Normal University, Changsha, 410081, China. E-mail: chenp@hunnu. edu.cn; [email protected]; [email protected]

These authors contributed equally to this work.

a Key laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, China b School of Biology, Hunan University, Changsha, China

Copyright © 2014 John Wiley & Sons, Ltd.

Combination of SPITC and 18O labeling reduces the need for extensive desalting by Zip Tip® chromatography (Millipore, Billerica, MA, USA) and generates a clearer spectrum with low background noise.[12,13] Because spontaneous loss of the 18 O atom in a normal aqueous environment affects the stability of 18O labeling as well as the experimental results, 18O labeling should be conducted after SPITC derivatization. The two-step reaction conditions were optimized using a standard peptide, and three peptides from a model protein digested with trypsin. Thus, both quantification and sequence information were acquired precisely and easily. To validate whether the combination of labeling methods is compatible and effectively simplifies quantification and identification processes, quantification information obtained from the MS/MS spectrum was compared with that from the MS spectrum, and data acquired from MS/MS spectrum matched well with the data acquired from MS spectrum precisely. Our findings collectively confirm the utility of this method, both for standard samples and crude venom from a species of spider lacking genomic information.

In-solution digestion of samples Solutions of samples (BSA, spider toxin and spider crude venom) were prepared by dissolving each sample in 50 mM ammonium bicarbonate buffer. Enzymatic digestions of samples were performed using sequencing-grade modified trypsin after thermal denaturation, as described elsewhere.[22] Briefly, each protein sample dissolved in 50 mM ammonium bicarbonate buffer was denatured thermally at 100 °C for 5 min. The denaturation process was quenched by placing samples in a freezer, and then the samples were reduced and alkylated with DTT and iodoacetamide at 10 mM and 55 mM concentration, respectively. The denatured proteins were digested in-solution by the addition of trypsin (1 : 50 protease/protein) and incubation of the reaction mixture overnight at 37 °C. Finally, the peptide samples were completely dried in a Speed-Vac (Thermo Savant, NY, USA) and kept at 20 °C until required for further experiments.

N-terminal derivatization of peptides with SPITC and trypsin-catalyzed 18O tagging

Experimental Chemicals 4-Sulphophenyl isothiocyanate, model peptide (EGVNDNEEGFFSAR, molecular weight: 1570 Da), bovine serum albumin (BSA), CCA and THAP and DAC were obtained from Sigma Aldrich (St. Louis, MO, USA). Trypsin was purchased from Promega Co. (Madison, WI, USA). C18μZipTipsTM were purchased from Millipore (Bedford, MA, USA). 18O water (Purity 97%) was obtained from Huayi Chemical Co., Ltd. (Jiangsu, China); all other chemicals were analytical reagent grade and used without further purification.

4-Sulfophenylisothiocyanate was prepared at a concentration of 2.55 mg/ml in NaHCO3 buffer (50 mM, pH 9.5). This solution was aspirated into contact with the bound sample on the ZipTip (making sure no bubbles were present), then left in the ZipTip and incubated at 55 °C. After 30 min, excess reagent was washed out with 0.1% trifluoroacetic acid (TFA) and the derivatized peptide(s) were eluted by 80% acetonitrile/0.5% TFA. The derivatized samples were divided equally into two parts and dried completely in a vacuum centrifuge. After resuspending in buffer (pH 6.86) and adding trypsin enzyme (trypsin : peptide = 100 : 1), the post-digestion labeling were carried out at 37 °C for 16 h.

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Figure 1. Comparison of mass spectrometry spectra of peptides using α-cyanohydroxycinnamic acid (CCA) or 2,4,6-trihydroxyacetophenone (THAP) matrix (desalted or no processing). (A) and (B), non-desalted sample using CCA matrix. (C) and (D), desalted sample using THAP matrix. (A2)–(D2), enlarged scale partly from (A) to (D).

W. Zhang et al. The buffer and enzyme were prepared by 18O-water or 16O-water, respectively. Labelled sample desalted by using C18 ZipTipsTM before MS analysis.

by the software provided by ExPASy (http://www.expasy.ch/ tools/blast/), using the default settings for MS BLAST.

Results and discussion

Preparation of MALDI matrices The CCA matrices were prepared as a saturated solution in 1% TFA in acetonitrile/water (1 : 1, v/v). The THAP matrix was prepared into a 10 mg/ml solution by dissolving 2′,4′,6′-trihydroxyacetophenone monohydrate in water/acetonitrile (50 : 50, v/v) followed by the addition of 50 mg/ml DAC (aq) at a ratio of 9 : 1 (v/v). Mass spectrometry and data processing All MS experiments were acquired on a Bruker Ultraflex (MALDITOF/TOF) mass spectrometer (Bruker Daltonics) equipped with a delayed extraction ion source. MS spectra were acquired in positive ion reflector mode with 400 laser shots per spot. Post source decay spectra were acquired with 3000 laser shots and 1 kV collision. Precursor ions were accelerated to 8 kV and selected in a timed ion gate. The fragments were accelerated by 19 kV in the LIFT cell. Precursor window selection was +/ 8 Da. External mass calibration was performed with a mixture of peptide standards (angiotensin I, m/z 1296.6853; ACTH fragment 1–17, m/z 2093.0867; ACTH fragment 18–39, m/z 2465.1989). Peptide quantification was performed by integrating and summing up corresponding isotope peak areas of peptides pairs (Flex-Analysis software). The de novo sequences were Blast

De novo sequencing of the model peptide In many cases, ionization suppression of SPITC-derivatized peptides hampers the quality of peptide sequencing, possibly as a result of salt contamination. Ionization suppression from contaminants often becomes a severe limiting factor in the identification of sulfonated peptides. A recent study showed that compared with CCA, matrix THAP/DAC enhances the response of sulfonated peptides by improving both the number and mass range of detected SPITC-derivatized peptides.[13] In our experiments, for a sample without desalting, the noise level in an MS spectrum obtained using CCA (Fig. 1(A1)) was significantly higher than that with THAP (Fig. 1(B1)) under the same laser level and frequency conditions, and consequently, low-abundance natural isotope peaks were covered with background noise. With a more sensitive scale, shown in Fig. 1(A2) and 1(C2), suggests that the resolution of natural isotope peaks is low assisted with CCA, which may automatically cause inaccurate calculation of the peak area. In comparison, spectra obtained with THAP as the matrix met the requirements for zero background noise and complete separation between natural isotope peaks. Desalting appeared to decrease the background noise and improve resolution of spectra with CCA (Fig. 1(A) and 1(C)). However, this did not

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Figure 2. Linear relativity of quantification in the ten times dynamic range for fibrin peptide. (A1)–(A8) show MS spectra of the fibrin peptide 18 before and after O labeling. The experimental ratios revealed that 0.46, 0.31, 0.25, 0.21, 0.128, 2.09, 2.98 and 4.03 correspond to specific theoretical ratios of 0.5, 0.33, 0.25, 0.2, 0.125, 2.00, 3.00 and 4.00, respectively. (B) Linear relativity graph in a ten times dynamic range of fibrin peptide 2 quantification, with R of 0.998.

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J. Mass Spectrom. 2014, 49, 400–408

Combination of SPITC and 18O labeling improve the detection of low-abundance peptides. Notably, some low-abundance peptides may have been lost upon desalting in spectra obtained using both CCA and THAP (Fig. 1 (B1), 1(C1) and 1(D1)). Matrix THAP containing DAC was a better choice for peptides subjected to SPITC/18O labeling, and therefore used for all subsequent analyses. In addition, the effect of labeling order was evaluated using a fibrin peptide. Neither SPITC derivatization followed by 18O labeling nor 18O labeling followed by SPITC derivatization had an observable effect on identification, but quantification was significantly affected because of spontaneous loss of the 18O atom in a normal water environment (data not shown). Thus, we conclude that 18O labeling should be conducted after SPITC labeling. Evaluation of the linear relativity of quantification on model peptides Quantification reliability was tested after de novo sequencing of the fibrin peptide. 18O and 16O labeling were performed in parallel after SPITC derivatization. Next, peptides labeled with 18 O were mixed with those labeled with 16O at a specific ratio (1/10 to 10/1), prior to MS/MS analysis. Figure 2(A1–A8) represent MS/MS spectra obtained upon spotting according to the following ratios: 1/2, 1/3, 1/4, 1/5, 1/8, 2/1, 3/1 and 4/1 (16O/18O, v/v). The results showed a favorable signal-to-noise ratio and good resolution. Through calculation of the peak area in the MS/MS spectrum, theoretical/experimental ratios of 0.5/0.46, 0.33/0.31, 0.25/0.25, 0.20/0.21, 0.125/0.128, 2.00/2.09, 3.00/2.98 and 4.00/4.03 were obtained, respectively. Experimental results achieved high consistency with theoretical data. Linear relativity of quantification in the ten times dynamic range was shown using curve-fitting software (Fig. 2(B)). The curvilinear equation was y = 1.028x + 0.016,

and R2 value was 0.998, indicating that the linear relativity of quantification is reliable. To investigate the effect of the tryptic procedure experiment on labeling stability, the method was further tested in tryptic peptides of BSA. As shown in Fig. 3(A) and 3(B), the R2 values of peptides 927, 1479 and 1567 Da were 0.938, 0.998 and 0.941, respectively, in accordance with the theoretical values. The linear relativity of quantification was reliable, suggesting that the tryptic procedure has no evident influence on the stability of labeling. Moreover, to investigate the effect of 18O labeling on MS/MS de novo sequencing, 16O/18O-labeled peptide pairs, 1142/1146, 1694/1698 and 1782/1786, from SPITC-derivatized peptides (927 + 215, 1479 + 215 and 1567 + 215) were separately sequenced in MS/MS analysis. As shown in Fig. 3(C–E), MS/MS spectra of peptides with 18O labeling (bottom) and without labeling (top) were similar, indicating no changes in the sequencing. The de novo sequencing results of peptides 927, 1479, 1567 Da, either 18O-labeled or unlabeled were YLYEIAR, LGEYGFQNALIVR and DAFLGSFLYEYSR, respectively. These findings confirmed that 18O labeling does not affect MS/MS de novo sequencing. Quantification and identification of peptides in a single MS/MS spectrum Usually, quantification and identification data are separately acquired from MS and MS/MS spectra. To simplify these processes, we focused on quantification information, which is generally overlooked in the MS/MS spectrum. The MS/MS spectrum of the SPITC/16O-derivatized peptide (m/z 1734) is presented in Fig. 4(A). MS/MS spectra of SPITC/16O-labeled peptide (m/z 1734) mixed with SPITC/18O

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Figure 3. Sequencing test in tryptic bovine serum albumin (BSA). (A) Mass spectrometry (MS) spectra of tryptic peptides from BSA (marked peaks indicate 927, 1497 and 1567 Da peptides). (B) Graph representing quantification linear relativity assessment of three tryptic peptides from BSA. (C)–(E) MS/MS spectra of labeled peptide pairs m/z 1142/1146, m/z 1694/1698 and m/z 1782/1786, respectively.

W. Zhang et al. labeled peptide (m/z 1738) at a 1 : 1 ratio are shown in Fig. 4(B). These two figures exhibited almost identical complete y-ion series with the same predicted sequence (compare Fig. 4(A) and 4(B)). The insert in Fig. 4(A) depicts an enlarged view of the MS/ MS spectrum of precursor peaks in A (m/z 1734) and B (m/z 1734/1738). As can be seen, we could not acquire quantification information based on Fig. 4(A). However, both quantification and identification data were evaluated from Fig. 4(B). Furthermore, the experimental quantitative ratio from the precursor peak (m/z 1734/1738) corresponded well with the theoretical ratio (1 : 1). An enlarged view of the MS/MS spectrum of y-ion series fragments is shown in the insert of Fig. 4 (B). From all the observed masses in the spectra, besides amino acid sequences, quantitative information could be

obtained from every fragment peak. However, fragment peaks did not correctly reflect the quantitative ratio. Thus, accurate quantitative information can be accessed from precursor peaks only, rather than the fragment ion peaks. To explore whether quantification information extracted from the MS/MS spectrum corresponds with that from the MS spectrum, SPITC/18O double-labeled peptides were mixed with SPITC/16O-labeled peptides at different 16O/18O theoretical ratios of 1/5, 1/3, 1/1, 3/1 and 5/1, respectively. For each ratio, quantification spectra both from MS/MS and MS spectra were similar. As shown in Fig. 4(C) and Table 1, MS/MS quantification data agree with the theoretical ratio, similar to MS data. Hence, protein identification and quantification was simultaneously achieved from a single MS/MS spectrum.

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Figure 4. Quantification and identification data were analyzed from a single mass spectrometry (MS)/MS spectrum. (A) MS/MS spectrum of tryptic 18 peptide after 4-sulphophenyl isothiocyanate (SPITC) derivatization, and (B) MS/MS spectrum of tryptic peptide after SPITC derivatization and O labeling. (C) Comparison of quantitative information obtained from MS and MS/MS spectra of a tryptic peptide.

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Combination of SPITC and 18O labeling This method was further tested in a mixed sample of spider toxins, HWTX- (ACKGVFDACTPGK NECCPNRVCSDKHKWCKWKL), HNTX-Ш (GCKGFGDSCTPGKNECCPNYACSSKHKWC KVYL) and JZTX-V (YCQKWMWTCDSKRACCEGLRCKLWCRKII), purified from three types of spiders, with the aim of identifying a protein/ peptide from a species with an unknown genome. To date, no genome database has been set up for these spiders. The three sequences of spider toxins were obtained in our laboratory using Edman sequencing.[23–25] To evaluate the dynamic range and feasibility of the quantitative assay with this double-labeled approach for the spider sample, HWTX- , HNTX-Ш and JZTX-V toxins were mixed at ratios of 2.0/3.0/5.0 (mixture 1) or 1.0/4.0/3.0 (mixture 2) (mol/mol), digested with trypsin, and labeled with SPITC and 16O/18O, respectively, as described earlier. Sample 1(16O) and Sample 2 (18O) were mixed at a 1 : 1 ratio for MS analysis, meaning that the theoretical ratios in HWTX- , HNTX-Ш and JZTX-V should be 2/1, 3/4 and 5/3, respectively. Analysis results for three types of spider toxins are shown in Table 2. The corresponding sequences for the 994, 968 and 1056 Da peptides were GVFDACTPGK, GFGDSCTPGK, and WMWTCDSER, respectively, and MS/MS analyses led to confident peptide identification for HWTX- , HNTX-Ш and JZTX-V. These sequences were confirmed in the spider toxin database. Meanwhile, experimental quantitative information with ratios of 1.4(HWTX-I), 0.76(HNTX-III) and 1.64 (JZTX-V) were matched practically with theoretical ratios of 2.0, 0.75 and 1.67, respectively. Application of the methodology for the venom from a single spider with an unknown genome This method showed good performance in the analysis of purified native peptides. We therefore wondered whether it might be employed to complex samples. Spider venom is a complex mixture containing proteins, peptides, organic compounds and salts. Studies indicated that spider venom is a rich source of components with therapeutic potentials and therefore attracts

Table 1. Quantification of MS and MS/MS spectra of double-labeled tryptic peptides 1734.8/1738.8 from BSA Theoretical ratio 16 18 of O/ O 1.00 0.33 0.20 3.00 5.00

Experimental ratio in MS spectrum

Experimental ratio in MS/MS spectrum

1.01 0.28 0.21 3.50 4.70

1.01 0.29 0.22 3.60 4.60

MS, mass spectrometry; BSA, bovine serum albumin.

attentions from scientists and pharmaceutical industry. Lines of evidences showed that different individual venom, such as venom from male and female spider species, contains different component composition or different abundance of the same component.[26,27] Elucidation of these differences might provide clues for investigating the mechanisms of spider toxin production and evolution. So far, no spider genome has been sequenced yet, and therefore, it is hard to identify and quantify spider venom components in a high-throughput manner. Therefore, this method has been further introduced for differential analysis of venom collected from single female or male spider Lycosa singoriensis.[28–30] Here, SPITC-modified and 16O/18O double-labeled tryptic peptides from the venom of female and male spiders were mixed at a 1 : 1 ratio, then identified and quantified to determine if any significant differences could be found between female and male spider venom. As shown in Fig. 5(A) and Table 3, tryptic peptide fragments from venom of male and female spiders have similar complexity. However, a number of peaks showed different abundance, reflecting biological differences between male and female spiders. Several peptide pairs with obvious differences were subjected to amino acid de novo sequence analysis. The corresponding MS/MS spectra of m/z 1205/1209 pairs and m/z 1453/1457 pairs, respectively, are shown in Fig. 5(B) and 5(C). The spectra mainly reveal y-ion series with excellent S/N. The fragment ion peaks of the 16O/SPITC double-labeled peptide (m/z 1205) represent the complete y1 to y7 y-ion series at m/z 174.5, 321.5, 408.4, 536.5, 641.4, 744.5, 877.7, 989.9 plus SPITC-labeled precursor ion 1205 (989.9 + 215 Da), thus facilitating interpretation of the CDMCQSF amino acid sequence by manual calculation of the differences between the adjacent y-ion fragments (Fig. 5(B)). Similar satisfactory results were achieved with the 18O/SPITC double-labeled peptide. As shown in 5C, 18O-labeled peptide m/z 1457 (1453 + 4 Da) yielded y-type fragment ions at m/z 178.5, 277.4, 390.3, 505.2, 602.3, 715.3, 798.2, 968.1, 1153.6 and 1242.6 (precursor ion), which corresponds well with the y1 to y10 sequence of SWAVSI/LPDILVR. In earlier studies, we observed a high-intensity signal in MS/MS spectra when proline existed, which was confirmed in this sequence (Fig. 5(B)). The mass gap between y4 (505.2 Da) and y5 (602.3 Da) was 79.1, indicating the existence of proline and y5 showed a very high-intensity signal. This intense signal in MS/MS spectra indicating the existence of proline further improved the accuracy of sequencing. There is currently no genome database for spider Lycosa singoriensis. For organisms with an unknown genome, homology search using the BLAST software is necessary.[31] Among all the possible sequences, we observed high identity of CDMCQSFR with rapidly accelerated fibrosarcoma (RAF) proto-oncogene serine/ threonine protein kinase from Myotis davidii.[32] The other BLAST results were listed in Table 3. Our findings present a clear target

Table 2. Quantification and sequencing data of tryptic peptides from spider toxin samples Name

MW unlabeled

HWTX-I HNTX-III JZTX-V

994 Da 968 Da 1056 Da

MW

16

18

16

O/ O labeled

1209 Da/1213 Da 1183 Da/1187 Da 1428 Da/1432 Da

18

Ratio( O/ O)

Sequence

Theoretical

Experimental

2.0 0.75 1.67

1.4 ± 0.57 0.76 ± 0.11 1.64 ± 0.23

GVFDACTPGK GFGDSCTPGK WMWTCDSK

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MW, molecular weight.

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Figure 5. Quantification and sequencing of tryptic peptides from spider (Lycosa singoriensis) venom. (A) Mass spectrometry (MS) spectrum of a tryptic 16 18 16 peptide mixture from male and female spider venom after 4-sulphophenyl isothiocyanate (SPITC) and O/ O labeling. (B)–(C) MS/MS spectra of O/ 18 SPITC-labeled peptide (m/z 1205) and O/SPITC-labeled peptide (m/z 1457), representing almost complete y-ion series with excellent signal-to-noise ratios.

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and provide direction for further research. An important point to note is that spider venom mainly contains small molecular mass peptides. One main advantage of SPITC modification is the

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215 Da increase in precursor ion mass, which promotes a shift of small peptides from the matrix cluster region (

A method combining SPITC and ¹⁸O labeling for simultaneous protein identification and relative quantification.

The relative quantification and identification of proteins by matrix-assisted laser desorption ionization time-of-flight MS is very important in /MS i...
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