Passive and Active Fragment Ion Mass Defect Labeling: Distinct

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Passive and Active Fragment Ion Mass Defect Labeling: Distinct Proteomics Potential of Iodine-Based Reagents Yu Shi, Bekim Bajrami, and Xudong Yao* Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269 The exact mass of a peptide differs characteristically from its nominal mass by a value called the mass defect. Limited by possible elemental compositions, the mass defect of peptides has a restricted range, resulting in an unoccupied mass spectral space in every mass-to-charge unit. The method of fragment ion mass defect labeling (FIMDL) places characteristic fragment ions of modified peptides as reporters into unused spectral space where no native peptide fragment ions exist. In this labeling method, peptides are chemically modified in solution and the modified peptides, upon gas-phase collision in a mass spectrometer, generate fragment ions with significantly shifted mass defects. In this work, the efficiency of iodine stable isotope-containing reagents for shifting mass defects of peptide fragment ions was systematically investigated, through derivatization of peptide N-termini with various reagents containing one or more chlorine, bromine, or iodine atoms. The observed efficiency for the iodine atom placing the labeled fragment ions into unoccupied spectral space agreed well with theoretical predictions from averagine-scaling analysis of ion masses. On the basis of the gas-phase stability of different labeling groups and their involvement in collisional dissociation of modified peptides, peptide modifications were classified into three categories: passive, type I active, and type II active. Each modification type has its unique potential in different proteome analyses. Possible proteomics applications of FIMDL are discussed and compared with proteome analyses currently being practiced in the field. Principles obtained from this survey study will provide a guideline in developing novel FIMDL reagents for advanced proteomics analysis. Peptide labeling is an important sample preparation method in mass spectrometry-based proteome analysis. Modification of peptide termini1-6 has been extensively explored, enabling global * Corresponding author. E-mail: [email protected]. (1) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Mol. Cell. Proteomics 2004, 3, 1154–1169. (2) Thompson, A.; Schafer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T.; Johnstone, R.; Mohammed, A. K.; Hamon, C. Anal. Chem. 2003, 75, 1895–904. (3) Liu, P.; Regnier, F. E. J. Proteome Res. 2002, 1, 443–450. (4) Regnier, F. E.; Julka, S. Proteomics 2006, 6, 3968–3979.

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analysis of proteome digests. While the peptide C-terminus can be advantageously labeled with stable oxygen isotope using enzyme catalysis,5,6 the peptide N-terminus is the focus of chemical modifications.1-4 The same reagents can also modify the amino group on the lysine side chain, however, which can be converted or protected before protein digestion. Alternatively, this predictable side-reaction can be taken care of during a protein database search via applying constraints for automated peptide sequencing. Chemical modification serves three major functions for peptide mass spectrometric analysis: (1) introducing quantification labels, (2) enhancing peptide spectral signals, or (3) simplifying spectral data complexity.7,8 However, the full potential of chemical labeling strategies is hindered by the fact that native peptide ions and chemical noise in the mass spectra interfere with mass spectrometric analysis of modified peptides and their fragments. An approach of fragment ion mass defect labeling (FIMDL) has been recently reported to address this common problem in chemical modification of peptides for mass spectrometry, together with a simple method of averagine-scaling analysis of mass spectral data.9 The scaling analysis uses the nominal mass of the hypothetical averagine residue10 as the mass reference, compared to the carbon-12 mass reference on the International Union of Pure and Applied Chemistry (IUPAC) scale.9 The mass defect of averagine residue is defined as zero.9 The FIMDL approach places the labeled peptide N-terminal fragment ions into unoccupied spectral space in mass spectra, via combining peptide modification in solution with gas-phase reaction of modified peptides. The mass shift of the modified fragment ions is due to large mass defects of atoms such as iodine in the FIMDL reagents. The usefulness of chlorine- and bromine-containing reagents for shifting labeled peptidyl ions into unoccupied spectral space have been previous demonstrated.11-13 Although iodine is also a halogen atom, and is thus conceivably useful for the same purpose, the effectiveness (5) Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836–2842. (6) Yao, X.; Afonso, C.; Fenselau, C. J. Proteome Res. 2003, 2, 147–152. (7) Leitner, A.; Lindner, W. Proteomics 2006, 6, 5418–5434. (8) Hung, C.-W.; Schlosser, A.; Wei, J.; Lehmann, W. D. Anal. Bioanal. Chem. 2007, 389, 1003–1016. (9) Yao, X.; Diego, P.; Ramos, A. A.; Shi, Y. Anal. Chem. 2008, 80, 7383– 7391. (10) Senko, M. W.; Beu, S. C.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1995, 6, 229–233. (11) Hernandez, H.; Niehauser, S.; Boltz, S. A.; Gawandi, V.; Phillips, R. S.; Amster, I. J. Anal. Chem. 2006, 78, 3417–3423. (12) Hall, M. P.; Ashrafi, S.; Obegi, I.; Petesch, R.; Peterson, J. N.; Schneider, L. V. J. Mass Spectrom. 2003, 38, 809–816. 10.1021/ac9008034 CCC: $40.75  2009 American Chemical Society Published on Web 06/29/2009

of iodine-containing reagents for separating modified fragment ions from native isobaric interference has only been recently demonstrated.9 The stability of iodine-containing compounds in solution and the gas phase was one concern. Therefore, it is important to experimentally examine the general usefulness of iodine-based reagents for producing and placing fragment ions in unoccupied spectral space. The radioactive iodine isotope (131I) has long been used as a tracer in biological and biomedical research. However, the stable iodine isotope (127I) has not been studied for the facilitation of peptide mass spectrometry until very recently.9 By comparison, chlorine and bromine have been used as mass defect labels for peptides, and labeled peptide molecular ions can be separated from native ions using high-resolution and highly accurate mass spectrometers.11 Chlorine- and brominecontaining peptide fragment ions, on the other hand, can be readily distinguished from other (nonhalogen containing) ions on mass spectrometers with unit resolution, based on detectable characteristic stable isotope distribution patterns of these ions. Chlorine and bromine atoms have two major stable isotopes. For mass spectra obtained on mass spectrometers of better-than-medium resolution and accuracy, a two-step algorithm has been established for filtering bromine-containing ions from native peptidyl ions. The first is based on the large but insufficient mass defect of the bromine atom and the second is based on the unique isotopic pattern.12 In contrast, iodine has only one stable isotope which does not add a signature isotope distribution to iodine-containing ions but rather introduces a larger mass defect. Iodine-containing ions are distinguishable based solely on mass separation. This separation can be successfully achieved for small ions (e.g., m/z 650) on mass spectrometers with better-than-medium mass resolution, e.g., time-of-flight mass spectrometers. With modern mass spectrometry instrumentation, the single stable isotope characteristic of the iodine atom can be advantageously used for improving the signal intensity and efficiency in using unoccupied mass spectral space.9 This work is a survey of a variety of chemical reagents possessing a halogen-substituted phenyl moiety, to shift the fragment masses into unoccupied mass spectral regions. Additionally, these reagents also possess a reactive group capable of modifying peptidyl amines via acylation,4 alkylation,14-19 carbamoylation,9,20 or thiocarbamoylation.9,21-26 These modifications encompass all known major methods of peptide amine derivati(13) Hall, M. P.; Schneider, L. V. Expert Rev. Proteomics 2004, 1, 421–431. (14) Hsu, J.-L.; Huang, S.-Y.; Chow, N.-H.; Chen, S.-H. Anal. Chem. 2003, 75, 6843–6852. (15) Fu, Q.; Li, L. Anal. Chem. 2005, 77, 7783–7795. (16) Hsu, J.-L.; Huang, S.-Y.; Shiea, J.-T.; Huang, W.-Y.; Chen, S.-H. J. Proteome Res. 2005, 4, 101–108. (17) Ji, C.; Guo, N.; Li, L. J. Proteome Res. 2005, 4, 2099–2108. (18) Russo, A.; Chandramouli, N.; Zhang, L.; Deng, H. J. Proteome Res. 2008, 7, 4178–4182. (19) Locke, S. J.; Leslie, A. D.; Melanson, J. E.; Pinto, D. M. Rapid Commun. Mass Spectrom. 2006, 20, 1525–1530. (20) Mason, D. E.; Liebler, D. C. J. Proteome Res. 2003, 2, 265–272. (21) Summerfield, S. G.; Bolgar, M. S.; Gaskell, S. J. J. Mass Spectrom. 1997, 32, 225–231. (22) Summerfield, S. G.; Steen, H.; O’Malley, M.; Gaskell, S. J. Int. J. Mass Spectrom. 1999, 188, 95–103. (23) Yalcin, T.; Gabryelski, W.; Li, L. J. Mass Spectrom. 1998, 33, 543–553. (24) van der Rest, G.; He, F.; Emmett, M. R.; Marshall, A. G.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 2001, 12, 288–295.

zation for mass spectrometry. This survey aims at identifying candidate compounds and more importantly underlying principles for further development of new FIMDL reagents. Tandem mass spectrometry (MS/MS) and energy-resolved mass spectrometry of modified peptides are compared. Modification reactions are categorized according to whether the modifying group is involved in gas-phase dissociation of modified peptide (active modification) or simply attached as an inert tag (passive modification). These two types of modification differ in the dependence on the collision energy (CE) for modified peptide ions generating N-terminal fragments. Importantly, each modification type has unique potential applications in proteome analysis. EXPERIMENTAL SECTION All peptides were purchased from AnaSpec (San Jose, CA). Reagents were purchased at better than reagent grade. Substituted benzaldehydes, benzoic acids, isocyanates, and isothiocyanates were purchased from either Sigma-Aldrich (St. Louis, MO) or Alfa Aesar (Ward Hill, MA). Formic acid (FA) was purchased from Fluka (Milwaukee, WI). Dimethyl sulfoxide (DMSO), dichloromethane (CH2Cl2), tetrahydrofuran (THF), and 4-(dimethylamino)pyridine (DMAP) were purchased from Acros Organics (Morris Plains, NJ). Trifluoroacetic acid (TFA) was purchased from Pierce (Rockford, IL). Horse apomyoglobin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) hydrochloride, 4-iodophenyl isocyanate, 2-picoline borane complex, (PICB), 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), N-hydroxysuccinimide (NHS), and iodoacetamide were purchased from Sigma-Aldrich (St. Louis, MO). Methanol, acetonitrile (ACN), acetic acid, dithioerythritol (DTE), and 2-mercaptoethanol (2-ME) were purchased from Fisher (Pittsburgh, PA). Trypsin (Roche Applied Sciences, Indianapolis, IN) was at sequence grade. Deionized water was purified by a Direct-Q water purifying system (Millipore, Billerica, MA). Drying of samples was performed either on SpeedVac (Savant, Farmingdale, NY) or on a lyophilyzer (Labconco, Kansas City, MO). High-performance liquid chromatography (HPLC) was performed on a 10ADvp system (Shimadzu, Columbia, MD) with solvent A of FA/ACN/H2O [2:10:988 (v/v/v)] and solvent B of FA/H2O/ACN [2:10:988 (v/v/v)]. Column temperature was set at 60 °C. Separations of peptides were carried out using a reversed-phase column (Hypersil GOLD, 1.9 µm, 100 mm × 1.0 mm, Thermo Scientific, Waltham, MA). Mass spectrometry was performed on either a quadrupole time-of-flight tandem mass spectrometer (QTOFmicro, Waters, Milford, MA), equipped with an in-house modified electrospray source, QSTAR Elite (Applied Biosystems, Foster City, CA), or 4000 QTRAP (Applied Biosystems, Foster City, CA). Preparation of Modified Peptides Using Substituted Benzaldehydes. To 10 µL of peptide (NSILTETLHR, designated as CFTR01) aqueous solution (1 nmol/µL), 10 µL of methanol, 5 µL of substituted benzaldehyde (200 mM) solution in methanol, 5 µL of acetic acid, and 10 µL of PICB (400 mM) in methanol were added individually. The reaction mixture (a total volume of 40 µL, 250 µM CFTR01, 25 mM aldehyde, and 100 mM PICB) was (25) Wang, D.; Kalume, D.; Pickart, C.; Pandey, A.; Cotter, R. J. Anal. Chem. 2006, 78, 3681–3687. (26) Wang, D.; Fang, S.; Wohlhueter, R. M. Anal. Chem. 2009, 81, 1893–1900.


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incubated at 50 °C for 2 h. A 700 µL of 0.1% TFA solution was added afterward to dilute the mixture, and the reaction was worked up by microscale CH2Cl2 extraction. The modified peptide in the aqueous phase was further purified using hydrophilic-lipophilic balance (HLB) materials (Waters) that was packed in-house in TopTips (10-200 µL, Glygen, Columbia, MD). A solution of 0.1% (v/v) TFA was used as the equilibration and washing solution and 70% (v/v) ACN as the elution solution. Preparation of Modified Peptides Using Substituted Benzoic Acids. To 10 µL of CFTR01 aqueous solution (1 nmol/µL), 40 µL of DMAP (300 mM) solution in THF, 60 µL of substituted benzoic acid (2 M) solution in DMSO, and 20 µL of EDC hydrochloride (1 M) in DMSO/H2O (4:1, v/v) were added individually. The reaction mixture (a total volume of 130 µL, 77 µM CFTR01, 92 mM DMAP, 923 mM acid, and 154 mM EDC) was incubated at 37 °C for 2 h. The workup procedures were similar as above. Preparation of 2,3,5-Triiodobenzoic Acid Modified Peptides. To prepare 2,3,5-triiodobenzoic acid succinamide ester, 2,3,5-triiodobenzoic acid (1 mmol), NHS (1.5 mmol), DMAP (0.2 mmol), and EDC hydrochloride (1.2 mmol) were dissolved in anhydrous dichloromethane (15 mL) and then stirred overnight at room temperature. The solvent was removed under vacuum, and the resulting solid was purified by column chromatography on silica gel using 35% ethyl acetate in hexane as the eluent. 1H NMR (CDCl3, 400 MHz): δ 8.46 (s, 1H), 8.12 (s, 1H), 2.94 (s, 4H). To 10 µL of CFTR01 aqueous solution (1 nmol/µL), 10 µL of triethylamine (400 mM) solution in DMSO and 100 µL of 2,3,5triiodobenzoic acid succinamide ester (200 mM) solution in DMSO were added individually. The reaction mixture (a total volume of 120 µL, 83 µM CFTR01, 33 mM triethylamine, 166 mM 2,3,5-triiodobenzoic acid succinamide ester) was incubated at 37 °C for 2 h. The workup procedures were similar as above. Preparation of Modified Peptides Using Substituted Isothiocyanates. To 10 µL of CFTR01 aqueous solution (1 nmol/ µL), 20 µL of substituted isothiocyanate (100 mM) solution in methanol/pyridine (1:1, v/v) and 20 µL of a mixture of methanol/ pyridine/H2O (1:1:1, v/v/v) were added individually. The reaction mixture (a total volume of 50 µL, 200 µM CFTR01, and 40 mM substituted isothiocyanate) was incubated at 45 °C for 30 min. The workup procedures were similar as above. Preparation of 4-Iodophenyl Isocyanate Modified Peptides. To 10 µL of CFTR01 aqueous solution (1 nmol/µL), 15 µL of HEPES (16.7 mM, pH 9) solution in water and 25 µL of 4-iodophenyl isocyanate (10 mM) solution in ACN were added. The reaction mixture (a total volume of 50 µL, 200 µM CFTR01, 5 mM HEPES, and 5 mM 4-iodophenyl isocyanate) was incubated at 25 °C for 1 h and was quenched with FA. The workup procedures were similar as above. LC-MS Analysis of Modified Peptides. Solutions of modified peptides (equivalent to 50 pmol of the native peptide as the starting material) were analyzed by LC-MS. Separation was run at 50 µL/min with a binary gradient: 1% B at 0 min f 1% B at 10 min f 70% B at 27 min f 70% B at 30 min f 1% B at 30.1 min f 1% B at 35 min. Key instrument parameters for mass spectrometer (QTOFmicro) were as follows: capillary voltage 3200 V, cone 6440


voltage (CV) 28 V, extraction voltage 1 V, collision energy (CE) offset 8 V. LC-MS/MS Analysis of Modified Peptides. MS/MS of modified peptides was run under a similar HPLC condition, and the doubly charged precursor ions were selected for the experiments on a QSTAR Elite mass spectrometer. Key instrument parameters were IS 5500 V, GS1 30, DP 80 V, CE 45 V, FP 250 V, DP2 15 V, and CAD 5. The spectra were processed with Analyst/ Bioanalyst 2.0 and internally calibrated with the immonium ion of His (m/z 110.0718), y1 (m/z 175.1195), y4 (m/z 526.3101), y6 (m/z 756.4004), and y7 (m/z 869.4844) ions. The calibrated spectra were further centroided with a signal threshold of 3 counts. The conversion of IUPAC exact mass (EM), nominal mass (NM, the round value of EMIUPAC), and mass defect (MD) of the peptide ions to those on the averagine scale was performed using equations described in a previous paper,9 which followed earlier equations used for studying ion mass defect.27,28 The MDAvg values were filtered by a lower cutoff threshold and an upper threshold, which were determined similarly.9 The ions that passed the filtering process were identified based on the theoretical EMIUPAC of N-terminal ions, and the intensities and m/z values of the passed ions were plotted using Origin software (V8.0773, B773; OriginLab Corporation, Northampton, MA). The unfiltered data were plotted similarly. EMAvg ) (EMIUPAC)0.999 493 894 NMAvg ) Round(EMAvg) MDAvg ) NMIUPAC - EMAvg Stability Study of Fragment Ions Generated from Modified Peptides. Modified peptides solutions (equivalent to 5 pmol/µL of the native peptide as the starting material, diluted by ACN/ 0.1% FA (1:1, v/v)) were infused (20 µL/min) through a syringe pump to a 4000 QTRAP mass spectrometer. Key instrument parameters were IS 5500 V, GS1 14, DP 120 V, IE1 1.8 V, IE3 0.6 V, and CAD 6. Six multiple reaction monitoring (MRM) transitions were selectively detected while ramping CE (5-130 V, step 1 V): [M + 2H]2+ f [M + 2H]2+, [M + 2H]2+ f b1, [M + 2H]2+ f a1, [M + 2H]2+ f FIMDL fragment ion (f), [M + 2H]2+ f y9, and [M + 2H]2+ f y7. LC-MS/MS Analysis of Modified Peptide Mixtures and Protein Digests. The solution with 1 pmol/µL concentration of six model peptides, laminin pentapeptide (YIGSR), YGR peptide (YGGFLR), desArg1-bradykinin (PPGFSPFR), CFTR03 (ISVISTGPTLQAR), osteocalcin (GAPVPYPDPLEPR), fibrinopeptide (ADSGEGDFLAEGGGVR), and CFTR02 (LSLVPDSEQGEAILPR), were prepared individually. The peptide mixture was prepared by mixing 20 µL of osteocalcin, 60 µL of laminin, 20 µL of YGR, 10 µL of CFTR02, 10 µL of CFTR03, 15 µL of bradykinin, and 15 µL of fibrinopeptide solutions. A solution of 10 µL of resulting peptide mixture was reacted with 4-iodobenzaldehyde, 3-iodobenzoic acid succinamide ester, and 4-iodophenyl isothiocyanate, individually, according to the above-mentioned procedures. Protein digests of lysozyme and (27) Kendrick, E. Anal. Chem. 1963, 35, 2146–2154. (28) Hughey, C. A.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G.; Qian, K. Anal. Chem. 2001, 73, 4676–4681.

Figure 1. Averagine-scale versus IUPAC-scale mass defect plots for elements. The solid dots are for mass defects on the averagine scale; the open dots are for the IUPAC scale.

myoglobin were produced by trypsin digestion, and resulting peptides were modified similarly. Theoretical Calculations of Averagine-Scale Mass Defect of N-Terminal Ions with Different FIMDL Groups. All theoretical fragment ions were generated using in-house scripts written in ActivePerl computer programming language. Monoisotopic residual mass of every amino acid residue was used for calculating the mass of peptide fragment ions. Fragment ions containing two amino acid residues were obtained by addition of monoisotopic residual masses of two residues and subtracting 26.987 09 Da for all a-ions, adding 1.007 825 for all b-ions, and adding 19.018 375 for all y-ions. In addition, the possibility of all fragment ions containing one 13C or two 13C were calculated by adding 1.003 355 for one 13C and 2.006 710 for two 13C, respectively. This Pearl script was developed in a loop style that calculated fragment ions containing up to eight amino acid residues, and the results generated were in the text format and contained the ion type, nominal mass, and amino acid sequence. Another script was developed to search for fragment ions (within the range of the nominal mass of interest) from the primary database. RESULTS Comparison of the Element Mass Defect on the Averagine Scale versus on the IUPAC Scale. The element mass defect was converted to the averagine scale (Figure 1), using a scaling factor of 0.999 493 894. Both MDAvg and MDIPUAC were plotted against the element nominal mass. The mass defect for MDAvg was more sensitive to the nominal mass increase. Iodine was located at the top of the plot. In comparison, the mass defect plot for MDIPUAC showed a plateau for a group of elements with similar mass defects. Mass Defect Plots for Peptide Fragment Ions with a Different Number of Iodine Atoms in the FIMDL Group. Mass defect plots were made for fragment ions with and without modification by iodine-containing benzoic acids (Figure 2A). Nominal masses and averagine-scale mass defects were calculated, and mass defect plots were made using Origin. Fragment ions appeared to be gathered into four different sets. The lowest set was composed of unmodified a-, b-, and y-ions (all in gray). The

other three sets included modified a- and b-fragment ions with an FIMDL group containing one, two, or three iodine atoms, respectively. The overlap between the labeled and native fragment ions decreased with the increase in the iodine number from 1 to 3; the crossing m/z value for the two groups of ions increased with the iodine number. Figure 2B-D showed the filtered MS/ MS spectra of peptide NSILTETLHR modified with NHS esters of mono-, di-, and triiodobenzoic acid, respectively. Chemical Modification of Peptides and Protein Digests with FIMDL Reagents. In general, modification of the peptide N-terminal amine was performed under standard peptide derivatization conditions, without extensive optimization. Thus, not every modification procedure resulted in quantitative conversion, which theoretically should be feasible. However, each modification produced a sufficient amount of modified peptides for mass spectrometric analysis, in order to examine the potential of a particular type of FIMDL reagents for proteome analysis. MS/ MS spectra of representative modifications were shown in Figure 3. Reductive alkylation of peptides with substituted benzaldehydes and o-phthaldialdehyde (OPA, AF8) was quantitatively achieved using PICB at elevated temperature (50 °C). It should be noted that only monoalkylated peptides were observed when modified with substituted benzaldehydes. Commonly used reducing reagent sodium cyanoborohydride (NaBH3CN) replacing PICB also gave high yields for peptide modification with benzaldehydes but not with OPA. The peptide modification using substituted benzoic acids was performed with EDC/DMAP in a mixture of DMSO and water. Incomplete modification was observed; the relative low reaction yield was due to the difficulty of finding a solvent system in which both peptides and halogen-substituted benzoic acids have good solubility. Two-step modification of peptides, via activating carboxylic acid by NHS first and then using the active NHS ester to modify the peptide, was found to be a technique of choice for improving modification yield. Modifying peptides with substituted phenyl isocyanates and isothiocyanates gave high conversion, following published procedures.9 These reagents were also used to modify standard peptide mixtures and protein digests. There were two general observations. One was that the population of doubly charged ions decreased compared with unmodified peptides, implying a decrease in the proton affinity after the peptide modification. This effect was much more significant for phenylcarbamoyl peptides than for alkylated peptides. The second observation was that the modified b1 ions and the complementary yn-1 ions were not always observed for phenylthiocarbamoyl peptides. In some cases, doubly charged yn-1 ions were observed instead, suggesting biased charge redistribution during the cleavage of the first N-terminal peptide bond. MS/MS spectra of selected peptides were shown in Figure 4. Averagine-Scale Mass Defect Filtering of MS/MS Spectra for Modified Peptides. The thresholds used in the averaginescale mass defect filtering were calculated similarly to a previously published procedure,9 based on the MDAvg distribution for the FIMDL fragment ions and the instrumental uncertainty in mass measurement. With the use of 3-iodobenzoic acid (PF3) modification as an example, the Gaussian fitting of the binned Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Figure 2. Mass defect plots and mass spectra for fragment ions with different numbers of iodine atoms. The mass defect plots (A) of native peptide fragment ions (a, b and y; in gray), and modified N-terminal ions (a and b) with benzoic acids containing one (red), two(green), and three (blue) iodine atoms. The MS/MS spectra of peptide NSILTETLHR modified with mono- (B), di- (C), and tri- (D) iodine-substituted benzoic acids, after averagine-scale mass defect filtering. * indicates the identified N-terminal ions with modifications.

(bin size 0.006 u) MDAvg values for the PF3-peptide fragment ions gave a σ value of 0.0316 u* and a peak value (xc) of 0.1861 ± 0.000 96 u* (the asterisk indicates the unit is on the averagine scale). The lower threshold was then calculated to be 0.1135 u*, the difference between xc and 2.3σ, and the upper threshold of 0.2588 u* to be the sum of xc and 2.3σ. The lower threshold value calculated from the Gaussian distribution was further reduced to 0.1037 u* by subtracting a value of 0.009 75 u* that was calculated for an ion with a mass of 650 u and an assumed statistical error of 15 ppm in mass determination (at a medium mass resolution, i.e., 5000). Accordingly, the upper threshold was increased to 0.2686 u* by adding a value of 0.009 75 u*. After the averagine-scale mass defect filtering, the MS/MS spectra of modified peptides were replotted using Origin. With an increase in the MDAvg for FIMDL groups (I3 > I2 > I > Br > Cl), the remaining fraction of interfering native peptide ions decreases (Figures 2 and 5). As expected, the majority of ions in the filtered spectra for the iodine- and bromine-containing labeling groups could be identified as N-terminal fragment ions with FIMDL groups. Calculation of Relative Collision Energy for Producing Fragment Ions from Modified Peptides. The generation and CID stability of fragments ions from peptides modified with different FIMDL reagents were studied using energy-resolved mass spectrometry in the MRM mode: from [M + 2H]2+ to the ion of interest. Signal intensities for transitions to six different ions, [M + 2H]2+, b1, a1, f, y9 (yn-1), and y7, were monitored, 6442


while CE offset was changed from 5 to 130 V at a step of 1 V. The correlation between the ion intensities and CE values was fitted as a single peak by an asymmetrical double sigmoid function using Origin. The CE value at 30% of the observed maximum ion intensity was read from the left halves of the CE profiles manually and designated as CE30 for each ion; 30% was arbitrarily chosen. If (CEa130 - CEb130) was negative, then a relative CE (CErel) was calculated to be (CEf30 - CEa130) for a peptide with one particular FIMDL group. Otherwise, CErel was calculated to be (CEf30 - CEb130). Results were summarized in Table 1. DISCUSSION Iodine-Containing Reagents for Fragment Ion Mass Defect Labeling of Peptides. FIMDL combines chemical modification of peptides in solution with gas-phase reaction in a mass spectrometer to generate fragment ions in unoccupied space in mass spectra. Fragment ions with FIMDL groups can be differentiated from isobaric native peptide fragment ions by averagine-scaling analysis, when the modifying groups have atoms of large mass defect like iodine. Iodine is one of the elements with the largest mass defect. On the averagine scale, this is more evident (Figure 1), due to (1) its large absolute mass defect and (2) relatively large atomic mass.9 Theoretical calculation indicates that mass separation of the labeled from native peptide fragment ions increases when the substitution on the FIMDL group

Figure 3. MS/MS spectra for peptide NSILTETLHR with N-terminal modifications. (A) Passive (3-iodobenzoic acid, PF3), (B) type I active (4-iodobenzaldehyde, AF3), and (C) type II active FIMDL (3-iodophenyl isothiocyanate, AF6).

changing from H to Cl to Br to I (Figure 5A.1-D.1) in peptides alkylated with substituted phenyl aldehydes. This trend in mass separation is also observed in filtered MS/MS spectra of modified peptides (Figure 5A.3-D.3), in which peak numbers decrease accordingly. It is interesting to note that although fragment ions with the bromine-containing FIMDL group have significant overlaps with native fragment ions on the theoretical averaginescale mass defect plot, the observed mass separation is about as sufficient as the iodine-containing fragment ions. This is due to the fact that not all of the theoretical interfering ions can be observed in a particular MS/MS spectrum. This suggests that

bromine-containing reagents might be useful for FMIDL as well. Compared to iodine-containing reagents, bromine-containing ones are generally easier to synthesize, but their potential for FIMDL is limited by the nearly equal abundance of two major isotopes, which results in a less efficient use of the m/z spectral space and decrease in the spectral signal intensity.9 Multiple iodine substitution on FIMDL groups increases the mass of modified peptides and the mass separation of fragment ions with the FIMDL groups from the native peptide ions (Figure 2). The increased mass separation on the averagine scale is attributed to the increase in both the absolute mass defect on the IUPAC scale and the mass of the labeling groups. For peptides acylated with mono, di, and triiodine-substituted benzoic acids, the mass separation increases for the same type of fragment ions (but with different numbers of iodine) from native ions. Therefore, the more iodine atoms on an FIMDL group, the less mass resolution and less mass accuracy a mass spectrometer needs to separate the labeled and unlabeled ions. Although triiodo labeling groups bring in higher mass defect, they are less desirable than diiodo ones, due to the potential overlap with triply charged ions in the same m/z unit. It was also observed that the increase in the number of iodine substitution led to a decrease in the yield of modified N-terminal fragment ions in the MS/MS analysis. Classification of Chemical Modification of Peptides with FIMDL Reagents. Peptide modifications with FIMDL reagents can be categorized into two groups: passive and active, according to whether the modifying group participates in peptide gas-phase fragmentation. The relative easiness for generation of fragment ions with FIMDL groups is the determinant used for classifying the modification type of a reagent. Depending on charge redistribution during the dissociation of multiply charged peptide precursors, fragment ions with FIMDL groups might be observed as shown in Figure 3. Passive FIMDL is defined for modifications that produce peptides capable of generating multiple N-terminal fragment ions with FIMDL groups upon gas-phase collision (Figure 3A). This implies that the FIMDL groups have similar gas-phase collision stability to other bonds, and modified peptides have minimal changes in the relative stability of peptide bonds. In MS/MS spectra of peptides with passive FIMDL, N-terminal ions with an iodine-containing labeling group can be readily filtered from native peptide fragment ions using scaling analysis.9 For peptides with active modification, FIMDL groups upon gasphase collision dissociate from modified peptides (type I, Figure 3B), or participate in gas-phase dissociation of neighboring peptide bonds (type II, Figure 3C). In other words, a particular bond in modified peptides is often more labile under gas-phase collision conditions than unaffected peptide bonds, as a result of active modification. Upon CID of peptides with the type II active FIMDL, the first N-terminal peptide bond dissociates preferentially. When terminal fragments with FIMDL groups carry a (positive) charge, they can be observed in MS/MS spectra; otherwise N-terminal fragments leave as neutrals. These observable N-terminal fragments include f, as well as a1 and b1 ions modified with FIMDL groups. There is a semiquantitative correlation between CErel calculated for a modified peptide (Table 1) and the modification type of FIMDL reagents. Passive FIMDL often has a medium CErel Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Figure 4. Representative MS/MS spectra for peptides with different sequences and different FIMDL groups. (A.1 and A.2) peptides modified by 3-iodobenzoic acid (PF3); (B.1 and B.2) peptides modified by 4-iodobenzaldehyde (AF3), and (C.1 and C.2) peptides modified by 4-iodophenyl isothiocyanate (AF7). * indicates the identified N-terminal ions with modifications.

value between 10-30 V. Type I active FIMDL is associated with small CErel values between 0-10 V, and type II active FIMDL has large CErel values larger than 30 V. 3-Pyridyl isothiocyanate (AF5) and OPA (AF8) were included in this study due to the commercial unavailability of their halogensubstituted analogues. The CErel values for AF5 and AF8 modified peptides may not be appropriate to be used to predict the FIMDL types of their halogen substituted analogues, because the structure difference may change the dependence on CE offset of modified peptides. However, halogen substitution does not significantly change the effect of FIMDL on peptide fragmentation for reagents used in this study. MS/ 6444


MS analysis of modified peptides suggests that AF5 and derivatives are type I, while AF8 and derivatives are type II active reagents. Although the use of CErel for modification typing is relative, it provides a useful tool to predict the relative easiness for generating fragment ions with FIMDL groups from modified peptides and to guide future design of new FIMDL reagents for proteomics applications. Passive Chemical Modification of Peptides. Modifications of peptide N-termini with halogen-containing benzoic acids, nicotinic acids, pyridinyl aldehydes and phenyl isocyanates belong to the passive FIMDL. After scaling analysis of MS/MS spectra, modified N-terminal fragment ions can be separated from native

Table 1. Collision Energy Obtained from Energy-Resolved Mass Spectrometry for Peptides with FIMDL Groups

peptide ions, if mass shift caused by FIMDL is sufficient. This typically requires that labeling reagents contain at least one iodine atom, in order to differentiate the labeled and native fragment ions by mass spectrometers with better-than-medium mass resolution. Averagine-scaling analysis can confidently identify fragment ions with FIMDL groups and thus readily deduce partial Nterminal sequences for peptides with passive FIMDL.9 Because these sequences are location-specific to the peptide N-terminus, they can be advantageously used as a constraint for precursor protein identification during a database search. A confidently defined short terminal sequence together with the precursor ion mass is in principle sufficient for identification of a peptide.29 Another potential application of passive FIMDL modification is associated with so-called parallel fragmentation mass spectrometry. When coeluting peptides have large differences in concentration, minor but important component peptides are often not selected for MS/MS analysis in common data-dependent or

information-dependent acquisition workflows. Therefore parallel fragmentation of peptides without precursor selection is proposed and implemented, aiming at improving the number of identified peptides and enhancing instrument duty cycle in bottom-up proteomic analysis. This method does not select particular precursors for MS/MS measurements, and all peptide ions are fragmented nonselectively, e.g., CID in the source region, in the collision cell, or in both places sequentially. In parallel fragmentation mass spectrometry, peptide precursors and their fragment ions are correlated after data acquisition, according to the peak elution time of the precursor and fragment ions.30 Clearly, without precursor selection, specific grouping of fragment ions is extremely important for deducing correct peptide sequence information. However, detection of sequence ions of minor peptides is often hindered by interfering isobaric fragment ions (through single and multiple bond dissociation) from major component peptides that coelute. The passive FIMDL of peptides generates sequence ions in unoccupied spectral spaces, free of interference.

(29) Nielsen, M. L.; Bennett, K. L.; Larsen, B.; Moniatte, M.; Mann, M. J. Proteome Res. 2002, 1, 63–71.

(30) Silva, J. C.; Gorenstein, M. V.; Li, G. Z.; Vissers, J. P.; Geromanos, S. J. Mol. Cell. Proteomics 2006, 5, 144–156.


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Figure 5. Averagine-scale mass defect plot and MS/MS spectra of peptide NSILTETLHR alkylated with ring-substituted phenylaldehydes. Averagine-scale mass defect plot of benzaldehyde (A.1), chlorobenzaldehyde (B.1), bromobenzaldehyde (C.1), and iodobenzaldehyde (D.1) modified peptide fragment ions, in which gray dots are for native peptide fragment ions, red dots for a-type ions, and blue dots for b-type ions. Unfiltered (A.2) and filtered (A.3) MS/MS spectra of benzaldehyde modified NSILTETLHR. Unfiltered (B.2) and filtered (B.3) MS/MS spectra of 4-chlorobenzaldehyde (AF1) modified NSILTETLHR. Unfiltered (C.2) and filtered (C.3) MS/MS spectra of 4-bromobenzaldehyde (AF2) modified NSILTETLHR. Unfiltered (D.2) and filtered (D.3) MS/MS mass spectra of 4-iodobenzaldehyde (AF3) modified NSILTETLHR.

This labeling method in combination with scaling analysis is expected to reduce this difficulty of parallel fragment mass spectrometry of peptides. A third proteomic application of passive FIMDL modification of peptides is that a series of observable fragment ions with the FIMDL group can be used as in-spectrum calibration ions. Mass 6446


determination of these ions can reach maximally possible instrument specifications in mass accuracy and resolution for a particular mass spectrometer, because these fragment ions are free from isobaric interferences. In addition, passive chemical modification of peptide termini with non-FIMDL reagents have long been used for facilitating

peptide sequencing by mass spectrometry, e.g., in the strategy of differential isotope-labeling of peptide termini.31 When labeling reagents carry differential stable isotope labels, modified terminal ions show in doublets which facilitate sequencing of modified peptides. Type I Active Chemical Modification of Peptides. Type I active chemical modification produces peptides that upon gasphase collision dissociate a fragment from the modifying group with a relatively high yield. This type of modification includes reductive alkylation with phenyl aldehydes and thiocarbamoylation with pyridinyl isothiocyanates of peptide N-terminal amines. Dissociation characteristics of these modified peptides are analogous to the generation of marker ions specific to peptides with post-translational modification. For example, phosphoserine and phosphothreonine peptides can produce negatively charged phosphate and related ions upon CID.32 Phosphoryl fragment ions have larger mass defects than isobaric native peptide ions, representing natural examples of FIMDL. Recently, a method of combining solution modification with a gas-phase reaction in a mass spectrometer has shown efficient generation of a positive cyclophosphoramidate ion for phosphoserine peptides.33 These marker ions are expected to be useful in proteomewide verification of phosphopeptides that are identified in proteomic profiling experiments. One potential proteomics application of type I active FIMDL of peptides, importantly, is quantitation of peptides and proteins using MS/MS. MS/MS tags like isobaric tag for relative and absolute quantitation (iTRAQ) reagents, which can multiplex several samples for quantitative comparison in a single experiment, represent the best examples. Quantitation using iTRAQ-type reagents is performed in the MS/MS mode by monitoring signal intensities of fragment ions or reporter ions generated from isobaric modified peptide precursors. Chemical background noise for the reporter ions can be reduced by designing these fragment ions that can be located in quasi-quiet regions in MS/MS spectra. Taking a different strategy, FIMDL places peptide fragment ions in the mass forbidden zone and therefore have a greater potential for “interference-free” quantitation. Type II Active Chemical Modification of Peptides. Type II active modification of peptide N-termini is uniquely interesting because collisional activation of modified peptides induces a sitespecific fragmentation of the modified peptides at/around the amino acid that carries the modification. At the peptide Nterminus, type II active modification includes phenylthiocarbamoylation of peptides and reductive alkylation by OPA. Substituted phenylthiocarbamoyl peptides produce modified b1 and the complementary yn-1 ions upon gas-phase dissociation of the first N-terminal amide bond. It is important to note that these N-terminal modifications produce fragment ions at high yields. For example, the yield is about 90% for iodophenylthiocarbamoyl peptides (AF6 and AF7). This aspect of peptides with type II active modification can find applications in MRM-MS, in which a high yield is essential for generating particular (31) Muenchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047–4057. (32) Carr, S. A.; Annan, R. S.; Huddleston, M. J. Methods Enzymol. 2005, 405, 82–115. (33) Shi, Y.; Bajrami, B.; Morton, M.; Yao, X. Anal. Chem. 2008, 80, 7614– 7623.

fragment ions. MRM-MS is gaining an increasing importance in verification, validation, and quantitation of biomarkers in proteome samples, as well as in targeted analysis of pathway proteomes. FIMDL of the first amino acid fragment ions could further push the intrinsic quantitation limits of the method by placing fragment ions in “interference-free” regions in the mass spectra. In order to achieve this advantage, however, variations of MRM-MS need to be developed to differentiate ions with FIMDL groups from isobaric interferences; detection of the FIMDL ions needs to have sufficient mass accuracy. Alternatively, more than one iodine atom is needed for shifting the modified fragment ions further away from isobaric interfering ions for standard MRM-MS on triple quadrupole mass spectrometers. In addition, a signal gain in the fragment ions should not be at the expense of decreasing the precursor mass spectral signal, which is a common issue with peptide N-terminal modification. This should be considered in designing practical FIMDL reagents for proteome analysis. The second important characteristic for peptides with type II active modification is that these peptides produce fragment ions carrying the first N-terminal amino acid residue information. Therefore these fragment ions are location-specific to the peptide N-terminus. This information, when combined with the mass of a peptide molecular ion, can help peptide identification without the need for a partial peptide sequence,16,24 especially when the proteome size is small. This method can be attractive for identifying low-abundance peptides at the border of the detection limit for intact molecular ions. In this situation, generation of evenly distributed fragment ions is not desired because the fragment ions may no longer be detectable. If only one or two fragment ions (such as b1 and yn-1) are dominantly generated, detection of the fragment ions can still be feasible. However, identification of peptides using this strategy requires a highly specific identification of the N-terminal amino acid residue. An approach for realizing the high specificity is to use highresolution and high-accuracy mass spectrometers like Fourier transform mass spectrometry (FTMS) for mass measurements; for example, a combination of the intact peptide ion with its b1 ion has been used.24 The specific sequence location of the b1 ion provides a constraint in database searching for peptide identification. Although it is a sound principle for peptide identification, its application to MS data of medium mass resolution and accuracy can be problematic.16 The decreased accuracy in mass measurement results in the reduced confidence in the identification of the N-terminal amino acid. With type II active FIMDL modification, this lost confidence can be regained. Reductive alkylation of peptides with aliphatic aldehydes also falls in the type II active modification category and generates modified a1 ions.14-17 Small aliphatic aldehydes commonly produce bisalkylated products.14-17 In contrast, benzylaldehydes under conditions used only result in monoalkylated products; their modification of peptides belongs to type I active modification. OPA, however, successfully forms bisalkylated peptides; presumably the second alkylation reaction is facilitated by the increased effective concentration of the second aldehyde group in the intramolecular reaction.18,19 The OPA-modified peptides produce stable modified a1 ion at high yield. The phenyl ring of OPA could Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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potentially be substituted with iodine atoms to synthesize FIMDL reagents. CONCLUSION Taking the opportunity of developing new reagents for labeling peptide fragment ions with sufficient mass defect, this work presents a system for typing chemical modifications of peptides according to fragmentation characteristics of modified molecules. Modifications are classified into passive, type I active, and type II active FIMDL reactions. Typing of peptide modifications provides a clear guidance in selecting different classes of iodine-based chemical reagents for distinct applications in proteome analysis. Although this work focuses on modification of the peptide N-terminus with FIMDL groups, general principles for the modification typing can be broadly applied to peptide modifications at the peptide C-terminus and side chains, as well as at post-

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translational modification sites. New FIMDL reagents will be designed based on this study and synthesized for advanced proteome analysis in order to beneficially utilize unoccupied mass spectral space. ACKNOWLEDGMENT This work was supported by the University of Connecticut, the Cystic Fibrosis Foundation (Grant YAO07XX0), and the American Cancer Society (Grant IGR-06-002-01). Dr. Pascal Lapierre at the Biotech Bioservice Center of the University of Connecticut is acknowledged for ActivePerl scripts used, which will be reported separately. Received for review April 14, 2009. Accepted June 8, 2009. AC9008034

Passive and Active Fragment Ion Mass Defect Labeling: Distinct

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