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Maleic Anhydride Labeling-based Approach for Quantitative Proteomics and Successive Derivatization of Peptides Shanshan Tian, Shuzhen Zheng, Yanpu Han, Zhenchang Guo, Guijin Zhai, Xue Bai, Xi-Wen He, Enguo Fan, YuKui Zhang, and Kai Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01120 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 24, 2017
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Analytical Chemistry
Maleic Anhydride Labeling-based Approach for Quantitative Proteomics and Successive Derivatization of Peptides Shanshan Tian,† Shuzhen Zheng,‡ Yanpu Han, ‡ Zhenchang Guo,† Guijin Zhai,† Xue Bai,† ‡
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Xiwen He, Enguo Fan, §, Yukui Zhang, , Kai Zhang*,
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2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China ‡
Department of Chemistry, Nankai University, Tianjin 300071, China Institut für Biochemie und Molekularbiologie, Universität Freiburg, Stefan-Meier-Straße 17, Freiburg 79104, Germany ∥ Department of Microbiology and Parasitology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/School of Basic Medicine, Peking Union Medical College, Beijing 100005, China §
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National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 116023, China ABSTRACT: Chemical derivatization is a simple approach for stable-isotope covalent labeling of proteins in quantitative proteomics. Herein we describe the development of a novel maleyl-labeling-based approach for protein quantification. Under optimized conditions, maleic anhydride can serve as a highly-efficient reagent to label the amino groups of tryptic peptides. Furthermore, ‘click chemistry’was successfully applied to obtain the second modification of maleylated peptides via thiolMichael addition reaction. Accurate quantification was further achieved via the first or/and second step stable-isotope labeling in this study. Our data thus demonstrate that the maleyl-labeling-based method is simple, accurate and reliable for quantitative proteomics. The developed method not only enables an enhanced sequence coverage of proteins by improving the identification of small and hydrophilic peptides, but also enables a controllable, successive, second derivatization of labeled peptides or proteins, and therefore holds a very promising potential for in-depth analysis of protein structures and dynamics.
Stable-isotope labeling technique combined with mass spectrometry (MS) analysis has become a powerful tool for the characterization of abundance and stoichiometry of proteins in biological system.1-3 Chemical derivatization is a simple and practical approach for stable-isotope covalent labeling of proteins.4 Considering the chemical reactivity and wide distribution of lysine, it has been thought to be a favorable site for labeling proteins. Several reagents, such as formaldehyde5-8 and succinic anhydride,9,10 have been applied to lysine derivatization for profiling differential expression of proteins. To date, lysine derivatization-based quantitative proteomics has attracted much attention.5,10-14 In a variety of derivatization chemicals of lysine, maleic anhydride is a unique reagent for modification of proteins.15 Chemical structure of maleic anhydride allows to perform a bifunctional reactivity. Firstly it can react efficiently with amino group of peptides (N-terminus and ε-amino group of the lysine side-chain). Then the resulting maleimide group has promising potential for further chemical modification with thiols via ‘click chemistry’.16,17 Although lysine maleylation has been used to improve the properties of proteins in drug delivery,18 food science19 and biomaterials,20 it has not been reported as an approach for quantitative proteomics. Owing to the special chemical properties of maleic anhydride and commercially available stable isotope labeling reagents, it is possible that maleic anhydride could be developed as a novel reagent for quantitative proteomics.
In this study, we investigated the maleylation procedures of peptides and further developed a novel maleyl-labeling-based approach to quantify and label proteins. Firstly, various reaction conditions were optimized and the labeling efficiency was evaluated at peptide and protein level, respectively. Then we combined the light and heavy stable-isotope maleic anhydride labeling with MS analysis for protein quantification of whole cell lysates. Furthermore, we achieved the fast second derivatization of maleylated peptides under mild aqueous reaction condition via ‘click chemistry’. Finally, four-plex labeling for quantitative proteomics was performed by using stable isotope maleic anhydride and Lcysteines at two stages. Our results show that the maleylationbased quantitative method is accurate and reliable for quantitative analysis of proteins and holds a potential of rapid second derivatization for in-depth analysis of proteins.
■ EXPERIMENTAL SECTION Chemicals and Materials. Maleic anhydride (12C4H2O3, MA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stable isotope maleic anhydride (13C4H2O3) and L-cysteines (13C3D315NH4O2S, Cys) were purchased from Cambridge Isotope Laboratories Inc. (MA, USA). β-Mercaptoethanol (C2H6OS, β-Me) and propanethiol (Pro) were from J&K Scientific Ltd. (Beijing, China). β-Mercaptoethanol (C2D4OHSH) was purchased from C/D/N Isotopes Inc. (Quebec, Canada). RPMI-1640 medium and
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fetal calf serum were from Thermo Fisher Scientific (Waltham MA, USA). Synthetic peptides ARTKQTARK and DAPPPAAK were from Abgent (WuXi, China). Methanol, DMSO, sodium bicarbonate, L-cysteines, triethylamine and other chemicals were analytical grade from Sangon Biotech (Shanghai, China). The Derivatization of Peptides using Maleic Anhydride. The derivatization reaction of ε-amine group of lysine residues and N terminal of peptides was performed with maleic anhydride. In brief, 20 ug of peptide was dissolved in 50 ul of 100 mM NaHCO3. And 4 ul of 0.5 M solution of maleic anhydride in methanol was prepared immediately before usage and added to the peptide sample dropwise. The solution was left shaking for 2 h at room temperature, and then dried in vacuum concentrator for further derivatization or MS analysis. The Second Derivatization of Maleylated Peptides via Click Chemistry. The producing maleylated peptides were reacted with L-cysteines, β-mercaptoethanol, and propanethiol, respectively. As an example, maleylated peptides were dissolved in 200 mM Tris-HCl (pH 7.5). And then L-cysteines was dissolved in H2O and added slowly to the samples. The reaction was allowed to proceed 2 h at room temperature using a test tube mixer and then dried in vacuum concentrator and desalted by C18 ZipTip (Millipore Corporation, USA) prior to MS analysis. Sample Preparation for Mass Spectrometry. Hela cells were cultured with modified RPMI-1640 medium supplemented with 10% fetal calf serum in a humidified incubator with 5% CO2 at 37 °C. Cells were lysed using Cell Lysis Kit (Sangon Biotech, Shanghai, China, containing both lysis buffer and protease inhibitors) (Supporting Information). Then tryptic digestion of cell ly21 sates was performed as previously described. Briefly, the protein pellet was suspended in 50 mM NH4HCO3 (pH 8.5), and digested with trypsin (Promega, Madison, WI) at an enzyme-tosubstrate ratio of 1:50 (w/w) for 16 h at 37 °C. The tryptic peptides were reduced with 5 mM dithiothreitol at 50 °C for 30 min and then alkylated using 15 mM iodoacetamide at room temperature for 30 min in darkness. The reaction was terminated with 15 mM cysteine at room temperature for 30 min. To ensure the complete digestion, additional trypsin at an enzyme-to-substrate ratio of 1:100 (w/w) was added to the digestion, and the mixture was incubated for an additional 3 h. Then the samples were dried in vacuum concentrator. The lyophilized sample was re-dissolved with 100 mM NaHCO3 and divided into two equal volumes and then labeled with either heavy or light maleic anhydride reagent (13C4H2O3 or 12C4H2O3), respectively. The two labeled samples were mixed equally and dried in vacuum concentrator. In the case of quantitative analysis for whole cell lysates by second derivatization via click chemistry, identical methods were adopted. After labeling with maleic anhydride, the sample were divided into two equal volumes and dried. Following with dissolved with methanol, stable-isotope β-mercaptoethanol (C2H2D4OS or C2H6OS) were employed separately in two equal sample and trimethylamine were chosen as catalyst, then mixed equally. The mixed peptides were dried or separated on high-pH reversed-phase HPLC (Supporting Information). MALDI-TOF-MS Analysis. MALDI-TOF analysis was performed using Autoflex III TOF/TOF mass spectrometer (Bruker Daltoniccs, Leipzig, Germany). The measurements were carried out in reflex positive-ion mode with delayed ion extraction. Prior to analysis, the instrument was externally calibrated with a mixture of peptide standards. DHB was used as the matrix for the analysis of peptides. 1.0 µl of the sample aliquots were placed onto MALDI plate, and then 1.0 µl of the DHB matrix was added and dried at room temperature. MS data were analyzed using Flexanalysis software for spectral processing and peak detection.
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Nano-HPLC-MS Analysis and Sequence Database Searching. 5 ul of sample was injected into a Nano-LC system (EASYnLC 1000, Thermo Fisher Scientific, Waltham, MA). Peptides were separated by a C18 column (50 µm inner-diameter × 15 cm, 2 µm C18, Thermo Fisher Scientific, Waltham, MA) with a gradient HPLC at a flow rate of 200 nL/min (Supporting Information). The HPLC elute was electrosprayed directly into a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The spray voltage was set at 1.8 kV. The mass spectrometric analysis was carried out in a data-dependent mode with one full MS scan followed by 10 HCD scans. For full MS survey scan, automatic gain control (AGC) target was 3e6, scan range was from 350 to 1750 with the resolution of 70,000. The 10 most intense peaks with charge state 2 and above were selected for fragmentation by higher-energy collision dissociation (HCD) with normalized collision energy of 27%. The MS2 spectra were acquired with 17,500 resolution. The exclusion duration for the datadependant scan was 10 sec, the repeat count was 2, and the exclusion window was set at 2.2 Da. The resulting MS/MS data were searched against UniProt Human database (downloaded July, 9, 2014, 87,724 entries) using Proteome Discoverer software (Version 1.4) with an overall false discovery rate (FDR) for peptides of less than 1%. Database search of labeled lysozyme was restricted to tryptic peptides of lysozyme sequence FASTA file. Peptide sequences were searched using trypsin specificity and allowing a maximum of two missed cleavages. Mass tolerances for precursor ions were set at 10 ppm for precursor ions and 0.02 Da for MS/MS. Carbamidomethylation (+57.021 Da) on cysteine was set as fixed modification and methionine oxidation as variable modification was selected. For relatively quantitative analysis of modified peptides between light/heavy samples, light maleylation (+98.000 Da) and heavy maleylation (+102.014 Da) on N-terminal and lysine were set as variable modifications. As for the quantification of second derivatization, N-terminal maleylation/β-mercaptoethanol (+176.014 Da) or N-terminal maleylation/β-mercaptoethanol-d4 (+180.039 Da) were set as variable modifications. For four-plex labeling for quantitative proteomics via stable isotope maleic anhydride and L-cysteines, light maleylation/light L-cysteines (LL, +219.020 Da), heavy maleylation/light L-cysteines (HL, +223.034 Da), light maleylation/heavy L-cysteines (LH, +226.046 Da), heavy maleylation/heavy L-cysteines (HH, +230.059 Da) were set as variable modifications. The distributions of Heavy/Light ratios of quantified proteins and peptides were generated by Proteome Discoverer.
RESULTS AND DISCUSSION Analysis Strategy of Maleyl-Labeling. Chemical derivatization of N-termini and ε-amino groups of lysine residues is an effective approach to induce stable-isotope labeling for different biological samples. Succinic anhydride, as a stable-isotope label9 ing reagent, has been applied to quantitative proteomics for mon10 itoring protein conformational changes and dynamics and dis22 covering protein adducts. Compared with succinic anhydride, we observed that maleic anhydride had a comparable labeling efficiency (Figure 1 and Figure S1). Meanwhile, the reaction product holds a potential of the second derivatization via ‘click 16,17 chemistry’ for further analysis. So we developed a stableisotope maleyl-labeling method for quantitative analysis of proteins. The proposed method includes the following steps: (1) proteins is subjected to tryptic digestion; (2) both amine group of lysine side chain and N-terminal of peptides are labeled with light or heavy isotope maleic anhydride via a fast acylation reaction, respectively; (3) the second derivatization of maleylated peptides is performed via ‘click chemistry’ with reagents, respectively; (4)
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mix the equal amount of light and heavy labeled samples for MS analysis.
slightly higher than that with succinic anhydride in the optimized conditions. The optimized conditions were further employed to label Hela cell lysates, 3369 peptides (about 98%) were labeled in 3424 peptides corresponding 1216 proteins, which further indicates the feasibility of maleyl-labeling approach for the quantification of proteins and peptides.
Figure 2. MALDI-TOF-MS of peptide ARTKQTARK (1058.63 Da) and its labeling product (1353.77 Da) with maleic anhydride in different solvents: (A) control experiment (no reaction); (B) H2O; (C) NaHCO3; (D) DMSO; (E) CH3OH. Figure 1. (A) The strategy of succinic anhydride labeling. (B) The strategy of maleic anhydride labeling. (C) MALDI-TOF-MS of peptide ARTKQTARK (1058.63 Da), peptide labeled with maleic anhydride (1353.77 Da) and peptide labeled with succinic anhydride (1359.86 Da). Optimization and Characterization of Maleic Anhydride Labeling. To obtain a high efficiency of maleyl-labeling peptides, the derivatization reaction was optimized. A peptide ARTKQTARK (1058.63 Da, histone H3, 1-9 residues) was chosen for the reaction in different conditions. We first investigated the effect of solvents on derivatization. The complete product is found at 1353 Da (a mass shift of +294 Da by adding two maleylation on lysine and one on N-terminal), when CH3OH or DMSO as solvent, as shown in Figure 2. Considering the compatibility with MS analysis, CH3OH was determined as the solvent of maleic anhydride for following experiments. And then we studied the influence of ratio of peptide and maleic anhydride. MALDITOF-MS analysis (Figure 3) showed that a relatively complete labeling of the peptide was achieved when maleic anhydride was 100-fold molar excess. Therefore, we chose 100-fold excess as the optimized condition. Next, we compared the reaction efficiency at different reaction times. It can be seen from Figure 4 that the reaction is complete within 1 h, and there were no by-products when prolong the reaction time. Considering the complexity of the biology samples, we chose 2 h as the reaction time to obtain complete labeling. To test the robustness of maleyl-labeling, we used the optimized conditions for different peptides from the tryptic digestion of lysozyme and evaluated the results by LC-ESI-MS analysis. Figure 5 showed MS spectra of two typical peptides without or with maleylation. CELAAAMK (residues 4-17) and FESNFNTQATNR (residues 35-46) were completely labeled as expected mass shift in MS. Furthermore the comparisons of labeling efficiency between maleic anhydride and succinic anhydride were conducted in lysozyme digests. As shown in Table S1, the labeling efficiencies of the peptides with maleic anhydride are
Figure 3. MALDI mass spectra of the peptide ARTKQTARK (1058.63 Da) and its labeling product (1353.77 Da) with maleic anhydride in different molar ratio of maleic/amino: (A) control experiment (no reaction); (B) 10:1; (C) 50:1; (D) 100:1; (E) 200:1.
Figure 4. MALDI analysis of the peptide ARTKQTARK (1058.63 Da) and its labeling product (1353.77 Da) with maleic anhydride at different reaction time: (A) control experiment (no reaction); (B) 1 h; (C) 2 h; (D) 4 h.
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The Quantitative Analysis for Whole Cell Lysates using Stable-Isotope Maleyl-Labeling. To examine the reliability of the quantitative method for complex samples, we performed quantitative analysis of Hela cell lysates using maleyl-labeling-based method. As shown in Scheme S1 A, the whole cell lysates was digested with trypsin. The tryptic peptides were divided into two parts equally, and then labeled with the light and heavy maleic anhydride, respectively. After mixed with equal amount, the labeled mixture was subjected to HPLC-MS/MS analysis. As shown in Figure S2, 2846 peptides corresponding 1403 proteins were quantified. To check the reliability of this quantification approach, a paired t test was performed over all protein and peptide ratios. The t test showed no significant differences (p < 0.05) between our experimental data and the theoretical value (Figure S2). To check the details, we further analyzed the MS signal intensities of tryptic peptides labeled with light and heavy isotopes. Taking glucose-regulated protein for example, we observed a series of [M+2H]2+ precursor ions of maleylated peptides such as a pair of MA-VYEGERPLTKMA (heavy, m/z = 698.33, 2+ and light, m/z =694.32, 2+) (Figure 6). All H/L ratios are close to 1, which correlates with the theoretical value (1:1 mixed). We further calculated the determined ratio of H/L maleylation of 10 highest abundance of peptides from glucose-regulated protein (Table S2, MS/MS spectra of the typical maleylated peptides in Figure S3.). These results showed that this method is highly reliable and accurate for quantitative characterization of proteins between samples.
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Figure 6. MS spectra of peptides from Hela cell lysates labeled with light and heavy maleic anhydride. (A) MAVYEGERPLTKMA with heavy (m/z = 698.33, 2+) and light (m/z = 694.32, 2+). (B) MA-FEELNMDLFR with heavy (m/z = 708.32, 2+) and light (m/z = 706.31, 2+). (C) MADNHLLGTFDLTGIPPAPR with heavy (m/z = 1018.51, 2+) and light (m/z = 1016.51, 2+). Characterization and Optimization the Second Derivatization via Click Chemistry. Recently a fast thiol-quantification 16 assay has been developed. Inspired by the successful application of the click reaction, we developed the second derivatization on maleylated products. To evaluate the feasibility of the proposed method, we investigated the derivatization of maleylated peptides using three different reagents including β-mercaptoethanol, Lcysteines and propanethiol, respectively (Scheme 1). The reaction conditions were optimized using a standard peptide, respectively. Figure 4S A shows the labeling efficiency of L-cysteines at different ratios of peptides and reagents. A high labeling efficiency can be obtained when the ratio reaches to 100:1 (Figure 4S A). Our result showed that all three reagents enable to label maleylated product completely. This result demonstrated the second derivatization can be achieved.
Scheme 1. The second labeling for maleylated peptides via click chemistry.
Figure 5. ESI-MS of tryptic peptides CELAAAMK (lysozyme residues 4-17, (A)) and FESNFNTQATNR (lysozyme residues 35-46, (B)) with or without maleylation. Cysteine was alkylated before maleyl-labeling.
To examine the robust of the second derivatization, we further performed the two-step labeling of tryptic peptides of lysozyme. Taking peptide FESNFNTQATNR (m/z=1428.7) for instance, the peptide was labeled with maleic anhydride (MA) leading to a mass shift of 98 Da (Figure S4 C(b)). Subsequently, the second derivatization based on thiol-ene reaction with β-mercaptoethanol (β-Me) leading to an additional mass shift of 78 Da for the maleyled peptide (Figure S4 C(c)). The fragmentation behavior in HCD of the modified peptides illustrated the presence of each modification. Furthermore, we employed the two-step labeling to the whole cell lysates, and the corresponding derivatives of peptides were also obtained. Taking peptide QWYESHYALPLGR from RPS8 (m/z=1618.8) for example, we observed a series of [M+2H]2+ precursor ions of no labeling (m/z = 810.40, 2+), maleylation labeling (m/z = 859.40, 2+) and β-mercaptoethanol modification following with maleylation labeling (m/z = 898.41, 2+) (Figure 7). All the mass shifts caused by labeling were same to theoretical value. MS/MS spectra of the labeled peptide further validated the results. Interestingly, the sequence coverage of peptide mapping of lysozyme was increased after derivatization (Figure S5). After checking, we found the identification of short peptides were improved via the derivatization. Meanwhile the HPLC retention time of the MA peptides and MA-β-Me labeled peptides were both increased compared to that of the corresponding unmodified counterparts. It means that this two modifications could cause the increase of peptide hydrophobicity by neutralizing the charge of – NH2. The results were further confirmed in whole cell lysates
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(Figure S6). It indicates that the maleyl-labeling and second labeling enable to improve identification of small or highly hydrophilic peptides. All the above results demonstrated that the second derivatization was feasible. Therefore, we can introduce isotopes either in the first maleylation or the second derivatization, which hold a potential application for quantitative proteomics.
0.05) between our experimental data and the theoretical value 1 (1:1 mixed), which showed the reliability of the quantification method. In addition, we further analyzed the MS signal intensities of the peptides labeled with light and heavy isotopes. For example, a series of precursor ions of light and heavy isotopes modified peptides from pyruvate kinase PKM were identified, such as a pair of EAEAAIYHLQLFEELR (Figure 8B), RFDEILEASDGIMVAR and NTGIICTIGPASR (Figure S7). The result shows that the second-step “click-chemistry” based maleylation labeling is reliable and accurate for quantitative characterization of proteins between samples, which can expand the usage of the maleic anhydride labeling and make it more flexible and adaptable for multiplexed quantitative analysis in quantitative proteomics.
Figure 7. MS and MS/MS spectra of peptide QWYESHYALPLGR (m/z=1618.8) of no labeling (A, m/z = 810.40, 2+), maleylation labeling (B, m/z = 859.40, 2+) and βmercaptoethanol modification following with maleylation labeling (C, m/z = 898.41, 2+). The Quantitative Analysis for Whole Cell Lysates by Second Derivatization via Click Chemistry with Stable-isotope β-mercaptoethanol. To explore the feasibility of the rapid second derivatization for in-depth analysis of proteins, we also performed quantitative analysis of Hela cell lysates using stable-isotope βmercaptoethanol based on maleyl-labeling method (Scheme S1 B). After labeling with maleic anhydride, the sample were divided into two equal volumes and sequentially labeled with light and heavy stable-isotope β-mercaptoethanol, respectively. After mixed with equal amount, the sample was fractioned and analyzed by HPLC-MS/MS. The ratio of all the identified proteins and peptides were determined by PD and the results were shown in Figure 8A. As shown in Figure 8, 6462 peptides corresponding 2407 proteins were quantified. To check the reliability of this quantification approach, a paired t test was performed over all protein and peptide ratios. The t test showed no significant differences (p
