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Detailed map of oxidative post-translational modifications of human p21ras using Fourier transform mass spectrometry.

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  • 1. Mass Spectrometry Resource, Department of Biochemistry, Cardiovascular Proteomics Center, and Vascular Biology Unit, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
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    • Zhao C 1
    (1 author)

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Analytical Chemistry, 01 Jul 2006, 78(14):5134-5142
https://doi.org/10.1021/ac060525v PMID: 16841939 PMCID: PMC3098383

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Abstract 

P21ras, the translation product of the most commonly mutated oncogene, is a small guanine nucleotide exchange protein. Oxidant-induced post-translational modifications of p21ras including S-nitrosation and S-glutathiolation have been demonstrated to modulate its activity. Structural characterization of this protein is critical to further understanding of the biological functions of p21ras. In this study, high-resolution and high mass accuracy Fourier transform mass spectrometry was utilized to map, in detail, the post-translational modifications of p21ras (H-ras) exposed to oxidants by combining bottom-up and top-down techniques. For peroxynitrite-treated p21ras, five oxidized methionines, five nitrated tyrosines, and at least two oxidized cysteines (including C118) were identified by "bottom-up" analysis, and the major oxidative modification of C118, Cys118-SO3H, was confirmed by several tandem mass spectrometry experiments. Additionally, "top-down" analysis was conducted on p21ras S-glutathiolated by oxidized glutathione and identified C118 as the major site of glutathiolation among the four surface cysteines. The present study provides a paradigm for an effective and efficient method not only for mapping post-translational modifications of proteins but also for predicting the relative selectivity and specificity of oxidative post-translational modifications, especially using top-down analysis.

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Anal Chem. Author manuscript; available in PMC 2011 May 20.
Published in final edited form as:
PMCID: PMC3098383
NIHMSID: NIHMS63588
PMID: 16841939

A Detailed Map of Oxidative Post-translational Modifications of Human p21ras sing Fourier Transform Mass Spectrometry

Cheng Zhao,1,2 Mahadevan Sethuraman,2 Nicolas Clavreul,3 Parminder Kaur,1,2 Richard A. Cohen,2,3 and Peter B. O’Connorcorresponding author1,2
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Supplementary Materials

Abstract

P21ras, the translation product of the most commonly mutated oncogene, is a small guanine nucleotide exchange protein. Oxidant-induced post-translational modifications of p21ras including S-nitrosation and S-glutathiolation have been demonstrated to modulate its activity. Structural characterization of this protein is critical to further understanding of the biological functions of p21ras. In this study, high resolution and high mass accuracy Fourier Transform Mass Spectrometry (FTMS) was utilized to map, in detail, the post-translational modifications of p21ras (H-ras) exposed to oxidants by combining bottom-up and top-down techniques. For peroxynitrite-treated p21ras, five oxidized methionines, five nitrated tyrosines, and at least two oxidized cysteines (including C118) were identified by “bottom-up” analysis and the major oxidative modification of C118, Cys118-SO3H, was confirmed by several tandem mass spectrometry experiments. Additionally, “top-down” analysis was conducted on p21ras S-glutathiolated by oxidized glutathione and identified C118 as the major site of glutathiolation among the four surface cysteines. The present study provides a paradigm for an effective and efficient method not only for mapping post-translational modifications of proteins but also for predicting the relative selectivity and specificity of oxidative post-translational modifications especially using top-down analysis.

Introduction

The human p21ras (Ras) family consists of three frequently mutated proto-oncogenes, H-ras, K-ras, and N-ras, which encode ~21 kDa protein products.1, 2 The Ras proteins are small guanine nucleotide exchange proteins that play a critical role as signal transducers in the regulation of a number of cellular processes, including cell growth, differentiation, and apoptosis.1, 3 They cycle between inactive GDP-bound state and active GTP-bound state regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs).4–6 These regulatory proteins bind to Ras and regulate the exchange of GDP with GTP. Ras can be modified and activated by reactive nitrogen species (RNS) including nitric oxide (·NO), nitrogen dioxide (·NO2), dinitrogen trioxide (N2O3), and peroxynitrite 7 as well as reactive oxygen species (ROS) such as superoxide anion radical (O2−·) and hydrogen peroxide (H2O2).8–15 ·NO is the best characterized redox modulator of Ras activity as it has been shown to promote Ras GDP dissociation in vitro, GTP binding to Ras in vivo, and stimulation of signaling pathways downstream of Ras.16–22

Peroxynitrite anion (ONOO −), the product of the reaction between superoxide anion (O2−·) and nitric oxide (·NO),23, 24 is a potent oxidant formed in endothelial cells25, 26 which oxidizes a wide range of biological targets.23, 27, 28 Ras activity is potentially modulated by reversible as well as irreversible oxidative post-translational modifications. S-oxidation and S-nitrosation of Ras by peroxynitrite have been demonstrated in vitro and in vivo.13–15, 29 In particular, four surface cysteines (C118, C181, C184 and C186) with reactive thiol groups can regulate H-ras activity in response to oxidative modifications. The reaction of ROS/RNS with Cys-118, which is located in the Ras guanine nucleotide-binding motif (NKCD motif),30, 31 has been implicated in activation of Ras leading to downstream signaling to Raf-1/Mek/Erk to mediate cellular responses.7, 13, 32, 33 The role of oxidative modifications of the three terminal cysteines is unclear, but because they are involved in membrane localization of the protein,34–36 they could regulate the activation of Ras.

Glutathione (GSH), a tripeptide γ-glutamyl cysteinyl glycine, is oxidized by ROS/RNS to glutathione disulfide, GSSG. In mammalian cells GSH is the most abundant low molecular weight thiol, and thus it frequently binds to protein thiols to form mixed disulfides, a process termed S-glutathiolation. S-glutathiolated Ras (GSS-Ras) has been suggested to take part in cellular regulation.7, 12, 13

It has been generally accepted that S-oxidation, S-nitrosation, and S-glutathiolation of Ras is involved in cell signaling,37, 38 but the relative importance by which these modifications directly regulate activity remains poorly understood. The role of S-nitrosation of Ras C118 in mediating guanine nucleotide exchange and that of farnesylation of C186 and palmitoylation of C181 and C184 in mediating membrane localization has recently been studied by heteronuclear NMR and triple quadrupole mass spectrometry.39–41 However, the reports of structural characterization of post-translational modifications on full-length Ras protein are somewhat limited.41

Post-translational modification (PTM) mapping of biological samples is not only chemically and biologically challenging, but also technically demanding, particularly because some modifications may be at very low abundance and others are labile. The development of electrospray ionization (ESI)42 and matrix-assisted laser desorption ionization (MALDI)43, 44 techniques provide the soft ionization methods needed for mass spectrometric analysis of nonvolatile protein and peptide molecules. These two techniques, when combined with Fourier Transform mass spectrometry (FTMS), provide ideal instrumentation for structural analysis of PTMs due to superior resolving power and mass accuracy. Furthermore, FTMS is highly flexible with MS/MS experiments that utilize a variety of fragmentation techniques such as collision activated dissociation (CAD),45 multiple storage-assisted dissociation (MSAD),46, 47 infrared multiphoton dissociation (IRMPD),48 sustained off-resonance irradiation collision activation dissociation (SORI-CAD)49, 50 and electron-capture dissociation (ECD).51–53 In particular, ECD directs fragmentation to the peptide backbone and allows labile modifications to remain attached during the fragmentation process.54–57 In addition, a qQq front end has been combined with the ICR cell in FTMS.58, 59 In this hybrid design ions can be isolated in Q1 with separation windows less than 1 m/z unit59 which improves analysis of complex mixtures since the precursor ions can be isolated without HPLC separation. Another advantage of this design is that a low abundance ion after Q1 isolation can be accumulated for a longer time in Q2 to increase the quantity of that ion for better MS/MS spectra.

The high resolution and high accuracy of FTMS make “top-down analysis”,60, 61 a relative new technology, possible. In top-down analysis, the intact protein is directly fragmented without enzymatic digestion. Compared to the traditional “bottom-up analysis”, which involves enzymatic digestion first and then MS/MS analysis of the resulting peptides, the top-down analysis, by definition, achieves 100% sequence coverage and does not miss post-translational modifications. However, the number of inter-residue bond cleavages generated by doing top-down analysis varies with the efficiency of fragmentation, so that multiple sequence changes resulting in unchanged mass would not be detected unless there are cleavages between those modifications. Although it is highly demanding of instrumentation and sample preparation techniques and the resulting data are far more complex and difficult to analyze, top-down analysis is, without doubt, a valuable complementary technique to bottom-up analysis.

This study was focused on structural characterization of oxidant-induced post-translational modifications on p21ras using combined bottom-up and top-down techniques in order to generate a complete map of the post-translational modifications of p21ras. The oxidative modifications observed, including some very low abundance modifications of peroxynitrite-treated p21ras, were mapped in detail by bottom-up analysis and several modifications on Cys-118 were confirmed by CAD and ECD MS/MS fragmentation. Also, the number of glutathiolated cysteines in p21ras treated with oxidized glutathione disulfide (GSSG) was determined by the intact mass difference between glutathiolated p21ras and the control sample, and the major site of S-glutathiolation was identified by top-down analysis. This structural information will aid in understanding the function of p21ras.

Methods

Materials

Recombinant p21ras was obtained in buffer containing 20 mM Tris (pH 8.0), 45 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT) and 50% glycerol from BIOMOL (Plymouth Meeting, PA) and peroxynitrite was obtained from Calbiochem (San Diego, CA). GSSG and all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Trypsin was purchased from Promega (Madison, WI).

Sample preparation

Recombinant p21ras (10–20 µg) was dialyzed to remove commercial buffers components and treated for 5 min at 37°C with peroxynitrite (ONOO−, 100–250 µM) or with GSSG (100 µM) in phosphate buffer (0.1 M, pH 7.0). The control and treated samples were dialyzed against 100 mM ammonium bicarbonate buffer before used for FTMS experiments. For “bottom-up” analysis, the control and the modified samples were digested with trypsin at 1:25 (w/w) in 100 mM ammonium bicarbonate (pH 8.0) for 3 h at 37°C and purified with Poros 50 R1 solid phase extraction material (Applied Biosystems, Foster City, CA). For “top-down” analysis, the control and the modified samples were purified with Poros 50 R1 material.

FTMS instrumentation

A custom built qQq-FTMS with a nanospray source and 7T actively shielded magnet (Cryomagnetics Inc., Oak Ridge, TN) was utilized in this study.58, 59 The “qQq front-end” refers to a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2).62, 63 Ions can be isolated in the mass resolving Q1 and accumulated in Q2 prior to analysis in the ICR cell. The instrument was designed to employ several fragmentation methods, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD),45 Multipole Storage Assisted Dissociation (MSAD),46, 47 electron capture dissociation (ECD),51–53 infrared multiphoton dissociation (IRMPD),48 and sustained off-resonance irradiation (SORI) CAD.49, 50, 64 The front-end quadrupoles were controlled using the program LC2Tune 1.5 (MDS Sciex, South San Francisco, CA), and the program IonSpec99 (IonSpec, Lake Forest, California) controlled data acquisition in the ion cyclotron-resonance (ICR) cell. The purified and dried samples were dissolved in 50/50 acetonitrile/water containing 1% formic acid for the peptide samples and 0.1% trifluoroacetic acid for the protein samples to a concentration of ~1 pmol/µl solution. A ~2–3 µl sample was loaded into a glass capillary tip pulled with a micropipette puller (Model P-97, Sutter Instruments Co., Novato, CA) to ~1 µm orifice diameter and a stainless steel wire was inserted into the distal end of the tip to form the electrical connection. For CAD experiments, the parent ions were isolated by Q1, and were fragmented by low energy (15–30 eV) collisions with N2 gas in Q2. For ECD experiments, the parent ions were isolated in Q1 and externally accumulated in Q2. After being transmitted and trapped into the cylindrical ICR cell, ions were irradiated with ~0.2 eV electrons from a 1.2 A heated dispenser cathode65 for 15 to 300 ms and were fragmented. The total pulse duration for CAD and ECD experiments varied between 1000 ms and 2 s and all data were analyzed without apodization and with two zero-fills to improve mass accuracy. The standard ICR conditions used in this study were as same as those in prior publications.58, 59

Results and discussion

Unmodified p21ras

Figure 1A (left) shows the ESI spectrum of a sample of intact purified p21ras prepared in the presence of DTT. The resolved isotopic distributions of several high abundance peaks were fitted to model distribution (dots),66 the monoisotopic molecular weight (MI) of this sample was determined to be 21284.4032 Da, which is consistent with the theoretical mass of intact p21ras, 21284.5511 Da. This 7 ppm difference is within the expected error range of ~5–10 ppm for externally calibrated FTMS data. When comparing the mass of intact p21ras protein prepared in the absence (Figure 1B), or in the presence of DTT (Figure 1A (left)), a 2 Da mass difference was detected, suggesting the presence of a disulfide bond. Because the p21ras sample was purchased in buffer containing DTT, this disulfide bond probably re-formed after removal of the DTT by dialysis.

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A. (left) ESI spectrum of purified unmodified p21ras treated with DTT. (right) ESI spectrum of purified p21ras treated with peroxynitrite. Inset shows m/z 1185 – 1230. B. ESI spectrum of purified unmodified p21ras that is not treated with DTT.

In order to localize this disulfide bond, these two samples were digested using trypsin. A peptide spanning amino acids 170–185 including Cys 181 and Cys 184 was identified in both samples which was 2 Da lighter in the sample without DTT (Figure 2A, left) than in the sample treated with DTT (Figure 2A, right). Q2 CAD MS allowed localization of a disulfide bond to the two cysteines of the peptide (Figure 2B). This is the first report of a disulfide bond between C181 and C184 p21ras. However, since the p21ras used in this study was recombinant protein expressed in bacteria and C181 and C184 were not palmitoylated, this disulfide bond may not exist in intracellular situation.

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A. (left) ESI spectrum of peptide 170–185 including the Cys181 – Cys184 disulfide bond (2+). (right) ESI spectrum of this peptide (2+) after DTT treatment. B. CAD MS/MS spectrum of this peptide. Top shows observed cleavages with measured masses and predicted masses in parentheses.

Peroxynsitrite (PN) treated p21ras

The Ras sequence suggests that oxidative modifications could happen on many different sites such as methionines, cysteines, and tyrosines. The ESI spectrum of the intact purified p21ras treated with a 10-fold excess peroxynitrite is shown in Figure 1A (right). Due to the complex and heterogeneous oxidative modifications on p21ras, the spectrum still yielded a signal to noise ratio < 5 even after several steps of sample cleaning. The isotopic distribution of individual charge states cannot be resolved (inset), and apparently includes several overlapping distributions. The top-down method is very difficult under such conditions. Nozzle skimmer fragmentation was applied to all the charge states, but the data were too complicated to analyze. Thus, the bottom-up method was utilized to map the oxidative post-translational modifications on this sample. Fortunately, 100% sequence coverage was obtained, and Figure 3 shows the spectrum of the tryptic digest mixture. The peaks were labeled with the peptides positions. Those labeled with * indicate methionine oxidation modification, those labeled with Δ indicate both methionine oxidation and cysteine sulfonic modification, and those labeled with ○ indicate nitration on the peptides. The sequences of two peptides (6–16 and 17–42) (insets) were confirmed by CAD MS/MS data, and they were then utilized for internal calibration of the rest of the spectrum. Table 1 lists all the detected peaks after internal calibration. Most of the errors between experimental and theoretical mass of these peptides are within 2 ppm except several between 2 ppm and 3 ppm for peptides below ~S/N = 5. Overall, the average of the absolute value of the error in these peptides is 1.09 ±0.74 ppm.

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Spectrum of p21ras treated with peroxynitrite and digested with trypsin. The major peaks are labeled with peptide positions. Those labeled with 002A indicate Met-Ox, those labeled with [increment] indicate both Met-Os and Cys-SO3H, and those labeled with ○ indicate Tyr-NO2. Insets are the CAD MS/MS spectra of the unmodified peptides 6–16 and 17–42, which were used for internal m/z calibration.

Table 1

Peak list of peroxynitrite-treated p21ras tryptic digest.

PositionExp. Peak (in.)Charge StateM+H(In.)Theo. MassError(ppm)
1–16(1Met-Ox)541.965031623.87941623.8780−0.83
1–16(1Met-Ox)812.444021623.88021623.8780−1.34
1–16(1Met-Ox,1Nit)834.935021668.86221668.86310.55
6–16478.30102955.5942955.5940−0.18
6–16955.59301955.5930955.59401.05
17–41990.152032968.44042968.44060.07
17–42774.640043095.53653095.5326−1.27
17–421032.517033095.53543095.5326−0.89
17–42(1Nit)1047.509033140.51143140.51761.99
43–73(2Met-Ox)899.415043594.63653594.64021.02
74–88538.606031613.80241613.7998−1.58
74–88807.404021613.80021613.7998−0.23
74–1021148.575933443.71213443.71701.44
89–97597.782021194.55621194.5544−1.49
89–101564.951031692.83741692.8346−1.62
89–102462.990041848.93651848.9357−0.45
89–102616.983031848.93341848.93571.27
89–102924.972021848.93621848.9357−0.26
89–102(1Nit)631.980031893.92441893.9207−1.93
102–123(1Met-Ox)806.415032417.22942417.2281−0.52
102–123(Met-Ox,1CysO3)822.411032465.21742465.2128−1.85
103–117(1Met-Ox)544.617031631.83541631.8314−2.42
103–123749.051032245.13742245.1321−2.34
103–1231123.070022245.13222245.1321−0.03
103–123(1Met-Ox)566.038042261.12852261.1270−0.67
103–123(1Met-Ox)754.379032261.12142261.12702.50
103–123(1Met-Ox)1131.067022261.12622261.12700.36
103–123(1Met-Ox,1CysO)759.714032277.12642277.1219−1.95
103–123(Met-Ox, 1Cys(NO))764.377032291.11542291.12002.02
103–123(1Met-Ox,1cysO2)765.045032293.11942293.1168−1.11
103–123(1Met-Ox,1cysO3)578.034042309.11252309.1117−0.36
103–123(1Met-Ox,1cysO3)770.374532309.10792309.11171.67
103–123(1Met-Ox,1cysO3)1155.062022309.11622309.1117−1.94
103–117(1Met-Ox)816.420021631.83221631.8314−0.47
103–128(1Met-Ox)709.111042833.42052833.4188−0.61
103–128(1Met-Ox)945.145032833.41942833.4188−0.19
105–123673.327032017.96542017.96871.66
105–123(1Met-Ox)678.661032033.96742033.9636−1.84
105–123(1Met-Ox)1017.484022033.96022033.96361.68
129–135401.21502801.4222801.4219−0.34
129–135801.42301801.4230801.4219−1.37
136–147664.841021328.67421328.6738−0.28
136–147(1Nit)687.335021373.66221373.6589−2.38
148–161552.290531654.85591654.8553−0.33
148–161827.932021654.85621654.8553−0.53
148–161(1Nit)850.424021699.84021699.84040.13
150–161466.574031397.70641397.70650.11
150–161699.356021397.70421397.70651.66
150–1611397.703011397.70301397.70652.50
150–161(1Nit)721.851021442.69421442.6916−1.78
162–169540.326021079.64421079.6438−0.35
170–185(1Met-Ox)560.247531678.72691678.7239−1.76
170–185(1Met-Ox)839.867021678.72621678.7239−1.36
170–189688.980032064.92442064.9227−0.80
170–189(1Met-Ox)694.311032080.91742080.91760.12
170–189(1Met-Ox)1040.963022080.91822080.9176−0.28
170–189(1Met-Ox,1CysO3)710.307032128.90542128.9023−1.43
170–189(1Met-Ox,1CysO3)1064.957022128.90622128.9023−1.82
170–189(1Met-Ox,2CysO3)1088.949022176.89022176.8870−1.46
170–189(1Met-Ox,3CysO3)1112.940022224.87222224.8717−0.21
171–185(1Met-Ox)775.819021550.63021550.6289−0.82
171–189(1Met-Ox)976.916021952.82421952.8226−0.81
171–189(1Met-Ox,1CysO3)1000.907022000.80622000.80730.56

Error Average =1.09 σ = 0.74

Figure 4A plots the modifications detected in peroxynitrite-treated p21ras including irreversible methionine, tyrosine, and cysteine oxidations.67–72 Five methionines (M1, M67, M72, M111, M182) were oxidized and five tyrosines (Y4, Y40, Y96, Y137, Y157) were nitrated. Cys118, which resides in the “NKCD” loop, the GTP binding site, was oxidized. Cys118 sulfenic acid (SOH), sulfinic acid (SO2H), sulfonic acid (SO3H), and S-nitrosothiol (SNO) were all observed (Figure 5A) though those with sulfenic acid, sulfinic acid and nitrosothiol modifications were very low abundance. One possible reason for the low abundance S-nitrosation is that the nitrosothiol can be further oxidized to sulfonic acid.73 At least one of the three terminal cysteines (Cys 181, Cys184, and Cys186) was oxidized into Cys-SO3H. Peaks corresponding to two and three terminal cysteines with sulfonic modifications were also detected, but they were much lower intensity than the one with only one sulfonic modification. The abundances of the two primary oxidation products of the peptide 103–123 are shown in Figure 5A. Peptide 103–123 with a single oxidation is clearly the most intense. This peptide could be oxidized either at cysteine or methionine, but the CAD MS/MS data in Figure 5B confirmed that it is oxidized at Met 111, consistent with the higher rate of methionine oxidation over cysteine oxidation. Among the peaks of the peptide 103–123 with both methionine and cysteine modifications (Figure 5A and inset), the one at m/z 770.3 representing the peptide with both methionine oxidation and cysteine sulfonic oxidation is the strongest, consistent with either the higher rate of formation or the greater stability of sulfonic acid compared with sulfenic or sulfinic acid. This peak (m/z 770.3) was isolated by Q1 and fragmented in Q2 by low energy (15–30 eV) CAD (Figure 5C) as well as in the ICR cell by ECD (Figure 5D). Substantially better cleavage and coverage was observed in the ECD experiment than in the CAD experiment. The results are tabulated in table 2. Multiple b, y (b17–20 and y14, 15, 19 from CAD) and c, z (c17–20 and z15–18 from ECD) ions including both Met111 oxidation and Cys118 sulfonic modification were detected, and these ions demonstrated that the cysteine and methionine oxidative modifications can survive low energy CAD and ECD conditions. Interestingly, the CAD spectrum (Figure 5C) showed no evidence of sulfate loss, even though sequence coverage was extensive. In the ECD spectrum, both c (c9–20) and z (z13, 15–18) ions, which contain methionine, show adjacent oxygen-loss (at -15.9949 Da) peaks, indicating that oxidative modification on methionine can be lost under odd electron ECD conditions. Guan, etc.71 studied CAD and ECD of several Met (O)-containing peptides and they reported a characteristic methanesulfenic acid side chain lost (CH3SOH, 64Da) in CAD, which was also detected in our study.

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A. Map of oxidative post-translational modifications detected on peroxynitrite-treated p21ras. B: Detected modifications on glutathiolated- p21ras. The most abundant glutathiolation on C118 confirmed by top-down analysis is labeled with *. C: Top down map of the major singly glutathiolated p21ras. The cleavages labeled with & include C118 glutathiolation.

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A. m/z 745–775 from Figure 1B. T* = trypsin autodigestion B. CAD MS/MS spectrum from peptide 103–123 with Met111-Ox. C. CAD MS/MS spectrum from peptide 103–123 with both Met111-Ox and Cys118-SO3H modifications. D. ECD MS/MS spectrum from peptide 103–123 with both Met111-Ox and Cys118-SO3H modifications. Peptide sequences (right) show the observed cleavages.

S-Glutathiolated p21ras

Figure 6A shows the ESI spectrum of intact glutathiolated p21ras which shows at least three groups of charge state distributions. Compared to the ESI spectrum of unmodified p21ras in Figure 1B, the most intense charge state distribution, whose monoisotopic molecular weight (MI) was 21257.4387 Da (+18 charge state), indicated one glutathione addition to the p21ras and the other two indicated two and three glutathione additions, respectively. For the triply glutathiolated p21ras, the disulfide bond at Cys 181- Cys-184 was cleaved (MIp21ras +3SG – MIp21ras +2SG = MISG + 2Da).

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Top down spectra of p21ras. A. ESI spectrum of purified glutathiolated p21ras. B. CAD MS/MS spectrum and C. ECD MS/MS spectrum on isolated +19 singly glutathiolated p21ras. The peak labeled with * indicates electronic noise.

For the bottom-up experiments on tryptic peptides, all the cysteines, including the terminal three cysteines were glutathiolated (Figure 4B), although it is possible that some of them were formed by exchange of disulfide bonds between GSSG and p21ras protein/tryptic peptides during sample digestion. Although with bottom-up data, it is impossible to determine which cysteine is the major site of glutathiolation on p21ras, this difficulty is solved with top-down analysis, in which the specific intact modified protein ions can be selectively isolated and fragmented. The major glutathiolated p21ras ion (Figure 6A) was isolated by Q1 and then directly fragmented in Q2 by CAD and in the ICR cell by ECD and the resulting spectra are shown in Figure 6B and Figure 6C. Although only 64 of 188 inter-residue bonds were cleaved, the fragment ions provided enough information to localize the modification. The CAD spectrum in Figure 6B did not show any glutathiolation on the b112 fragment ion but b131 did include one glutathiolation indicating that a cysteine between amino acid 112 and 131 from the N-terminus was involved. Similarly, the ECD spectrum in Figure 6C, showed no glutathiolation at z38 but showed one at z79, indicating that the glutathiolation was on a cysteine between amino acid 38 and 76 from the C-terminus, thus identifying the site as C118. Therefore, these data identify C118 as the site of most abundant glutathiolation in GSSG-treated p21ras. Thirty-two cleavages (b6–9, b18–20, b29–31, b47, b51–55, b109, b112, b131, b137, b177, y31, y123, y126, y129, y131–133, y135–136, y179, y187) were obtained in CAD top-down analysis, 46 cleavages (c4–9, c11–27, c29–35, c37–38, c41, c46, c57, c68–69, c83–84, c87, z5–6, z11–12, z38, z76) were obtained in ECD top-down analysis, and 4 complementary pairs (b53 and y136, b54 and y135, c57 and y132, b177 and z12) were obtained, resulting in 100% sequence coverage on the singly glutathiolated p21ras. Also, the consecutive sequence tags such as c11–27 and c29–35 with ~2 ppm mass accuracy is more than sufficient to accurately assign the protein identity.

In order to further analyze Cys118-SSG, the 103–123 peptide with glutathiolated C118 was isolated by Q1 from the digestion mixture and fragmented using low energy (15–25 eV) CAD and a ECD MS/MS experiments (Figure 7A and B). C17–20, z8, z11–13, z15–19 and y6–12, y14–20 including Cys118-SSG were detected, confirming this modification. As before, the ECD experiment provided higher cleavage coverage than the CAD experiment, and most of the errors between experimental and theoretical mass of b, y and c, z ions were within 2 ppm (Table 3). During the ECD experiment, two glutathione losses were detected on z ions (z7-SG and z9-SG) while nine glutathiolated z ions (z8, z11–13, z15–19) were observed. The detailed results of these studies are plotted in Figure 4.

An external file that holds a picture, illustration, etc. Object name is nihms63588f7.jpg

A. CAD MS/MS spectrum of peptide 103–123 with Cys118-SSG modification. The inset is the ESI spectrum of the precursor ion. B. ECD MS/MS spectrum of this modified peptide. The peak labeled with * indicates electronic noise. The sequence at the top shows the observed cleavages.

Conclusions

This study combined bottom-up and top-down mass spectrometry methods on a recently developed FTMS instrument to study the post-translational modifications on the p21ras. Peroxynitrite-treated p21ras included multiple complex modifications that, despite their complexity, were analyzed using a bottom up MS/MS approach. Many oxidative modifications and some low abundance modifications such as Cys118-SNO, Cys118-SOH and 0nCys118-SO2H were identified. It is notable that the SNO modification was stable throughout digestion and MS analysis. Five oxidized methionines, five nitrated tyrosines, and at least two oxidized cysteines, including Cys-118 and one of the terminal cysteines, were identified. The structure of the most abundant oxidative modification on Cys-118, Cys118-SO3H, was confirmed by low energy CAD and ECD MS/MS experiments. Also, from top-down analysis, Cys-118 is identified as the major glutathiolated cysteine on p21ras. This top-down analysis was selectively performed on the specific singly glutathiolated p21ras using both CAD and ECD fragmentation, and the peptide 103–123 with the Cys118-SSG modification was isolated from the digests and fragmented to confirm this modification.

The detected mass accuracy of this FTMS instrument on either peptides or fragmentation ions is ~ 2 ppm with internal calibration except for some very weak peaks (signal/noise < 5) for which accuracy suffers due to noise distortion of the peak shapes. This high mass accuracy helps for both protein and peptide identification. In addition, this study provides a paradigm for an effective and efficient method for mapping the post-translational modifications of proteins. With appropriate software and search engines, this method also can be used to identify unknown proteins and peptides.

Supplementary Material

01

Table 2 (Supplemental data): Fragmentation peak list of peptide 103–123 with Met111-Ox and Cys118-SO3H modifications from Figure 5C, D.

Table 3 (Supplemental data): Fragmentation peak list of peptide 103–123 with Cys118-SSG modification from Figure 7A, B.

Acknowledgements

The authors would like to thank Jason J. Cournoyer, Vera Ivleva and Dr. Cheng Lin for the valuable suggestions. This research was supported by NIH P41-RR10888, N01-HV28178, R01 AG27080, P01 HL HL81587, MDS Sciex, the ACS Petroleum Research Fund, and an NIH Post-doctoral Research Fellowship T32 HL07224.

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