Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges

A kinetic model, based on the "mobile proton" model of peptide fragmentation, has been reported previously for quantitative prediction of low-energy collision-induced dissociation (CID) spectra of singly or doubly charged peptides. For peptides with three or more charges, however, the simulation process is complex and time-consuming. This paper describes a simplified model for quantitative prediction of CID spectra of peptide ions with three or more charges. Improvements on other aspects of the model were also made to accommodate large peptides. The performance of the simplified model was evaluated by generating predictions for many known highly charged peptides that were not included in the training data set. It was shown that the model is able to predict peptide CID spectra with reasonable accuracy in fragment ion intensities for highly charged peptide ions up to 5000 u in mass.     

Prediction of collision-induced dissociation spectra of common N-glycopeptides for glycoform identification

Confident identification of the glycan moieties in glycopeptides by collision-induced dissociation (CID) requires accurate prediction of the CID spectrum of the glycopeptides. In this Article, the kinetic model for the prediction of peptide CID spectra is extended to predict the CID spectra of N-glycopeptides. The model was trained with 1831 ion-trap CID spectra of N-glycopeptides and is able to predict ion-trap CID spectra with excellent accuracy in ion intensities for N-glycopeptides up to 8000 u in mass. A total of 524 common glycoforms including complex N-glycans with 2-4 antennas, plus high-mannose type and hybrid type, can be predicted.     

Comprehensive comparison of collision induced dissociation and electron transfer dissociation

Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique which has proven to be an excellent tool for the elucidation of labile post-translational modifications such as phosphorylation and O-GlcNAcylation of serine and threonine residues. However, unlike collision induced dissociation (CID), which has been studied for decades, the intricacies of ETD-based fragmentation have not yet been firmly established or systematically addressed. In this analysis, we have systematically compared the CID and ETD fragmentation patterns for the large majority of the peptides that do not contain such labile modifications. Using a standard 48 protein mix, we were able to measure false-positive rates for the experiments and also assess a large number of peptides for a detailed comparison of CID and ETD fragmentation pattern. Analysis of approximately 19,000 peptides derived from both standard proteins and complex protein samples revealed that (i) CID identified 50% more peptides than ETD; (ii) ETD resulted in approximately 20% increase in amino acid sequence coverage over CID; and (iii) combining CID and ETD fragmentation increased the sequence coverage for an average tryptic peptide to 92%. Interestingly, our analysis revealed that nearly 60% of all ETD-identified peptides carried two positive charges, which is in sharp contrast to what has been generally accepted. We also present a novel strategy for automatic validation of peptide assignments based on identification of a peptide by consecutive CID and ETD fragmentation in an alternating mode.     

Identification of sites of ubiquitination in proteins: a fourier transform ion cyclotron resonance mass spectrometry approach

Structural elucidation of posttranslationally modified peptides and proteins is of key importance in the understanding of an array of biological processes. Ubiquitination is a reversible modification that regulates many cellular functions. Consequences of ubiquitination depend on whether a single ubiquitin or polyubiquitin chain is added to the tagged protein. The lysine residue through which the polyubiquitin chain is formed is also critical for biological activity. Robust methods are therefore required to identify sites of ubiquitination modification, both in the target protein and in ubiquitin. Here, we demonstrate the suitability of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, in conjunction with activated ion electron capture dissociation (AI ECD) or infrared multiphoton dissociation (IRMPD), for the analysis of ubiquitinated proteins. Polyubiquitinated substrate protein GST-Ubc5 was generated in vitro. Tryptic digests of polyubiquitinated species contain modified peptides in which the ubiquitin C-terminal Gly-Gly residues are retained on the modified lysine residues. Direct infusion microelectrospray FT-ICR of the digest and comparison with an in silico digest enables identification of modified peptides and therefore sites of ubiquitination. Fifteen sites of ubiquitination were identified in GST-Ubc5 and four sites in ubiquitin. Assignments were confirmed by AI ECD or IRMPD. The Gly-Gly modification is stable and both tandem mass spectrometric techniques are suitable, providing extensive sequence coverage and retention of the modification on backbone fragments.     

Sequencing and mass profiling highly modified conotoxins using global reduction/alkylation followed by mass spectrometry

A novel high-throughput method for characterizing heavily modified peptides from cone snail venom is described. Unpurified cone snail duct venom, consisting primarily of multiply disulfide-bonded peptides, is reduced and alkylated using a global procedure in order to simultaneously reduce and derivatize dozens of disulfide-bonded peptides. Samples of Conus victoriae venom are analyzed by online liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-MS) with collisionally induced dissociation (CID). Comparison of the mass profiles of peptides and CID spectra before and after the global reduction and alkylation enables cysteine-containing conopeptides to be ascertained. In this case, over 40 conotoxins are characterized based on only two LC-ESI-MS experiments in terms of mass, number of disulfide-linked cysteine residues (and hence, potential toxin superfamilies), relative hydrophobicity, and other posttranslational modifications. Using this technique, over half of the amino acids (by mass) of several peptides are defined prior to any detailed sequencing studies. Further comparison of the mass data with previously published genetic information allows sequence verification of three novel peptides, termed vc5b, vc6b and vc6c, based on both LC-ESI-MS CID and nanoelectrospray ionization-ion trap-mass spectrometry (nanoESI-MS) experiments. This global method is ideally suited to the use of larger genetic databases in order to efficiently sequence peptides in Conus venoms and is also applicable to analysis of other disulfide-rich classes of peptides such as defensins, chemokines, and snake, spider, or other venoms.     

Improving de novo sequencing of peptides using a charged tag and C-terminal digestion

An improved method for peptide de novo sequencing by MALDI mass spectrometry is presented. The method couples a charge derivatization reaction with C-terminal digestion to modify tryptic peptides. The charge derivatization attaches a fixed charge group onto the N-termini of peptides, and the enzymatic digestion after the derivatization step removes C-terminal basic amino acid residues such as arginine and lysine. The fragmentation of the modified peptide(s) under low-energy CID conditions (MALDI Q-TOF mass spectrometer) yields a simplified yet complete ion series of the peptide sequence. The validity of the method is demonstrated by the results from several model protein digests, where peptide sequences were correctly deduced either manually or through an automated sequencing program.     

Data-dependent modulation of solid-phase extraction capillary electrophoresis for the analysis of complex peptide and phosphopeptide mixtures by tandem mass spectrometry: application to endothelial nitric oxide synthase.

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) of peptides in conjunction with automated sequence database searching of the resulting collision-induced dissociation (CID) spectra has become a powerful method for the identification of purified proteins or the components of protein mixtures. The success of the method is critically dependent on the manner by which the peptides are introduced into the mass spectrometer. In this report, we describe a capillary electrophoresis-based system for the automated, sensitive analysis of complex peptide mixtures. The system consists of an ESI-MS/MS instrument, a solid-phase extraction (SPE)-capillary zone electrophoresis (CZE) device for peptide concentration and separation, and an algorithm written in Instrument Control Language (ICL) which modulates the electrophoretic conditions in a data-dependent manner to optimize available time for the generation of high-quality CID spectra of peptides in complex samples. We demonstrate that the data-dependent modulation of the electric field significantly expands the analytical window for each peptide analyzed and that the sensitivity of the SPE-CZE technique is not noticeably altered by the procedure. By applying the technique to the analysis of in vivo phosphorylation sites of endothelial nitric oxide synthase (eNOS), we demonstrate the power of this system for the MS/MS analysis of minor peptide species in complex samples such as phosphopeptides generated by the proteolytic digestion of a large protein, eNOS, phosphorylated at low stoichiometry.     

Prediction of low-energy collision-induced dissociation spectra of peptides

A kinetic model, based on the "mobile proton" model of peptide fragmentation, was developed to quantitatively simulate the low-energy collision-induced dissociation (CID) spectra of peptides dissociated in a quadrupole ion trap mass spectrometer. The model includes most fragmentation pathways described in the literature, plus some additional pathways based on the author's observations. The model was trained by optimizing parameters within the model for predictions of CID spectra of known peptides. A best set of parameters was optimized to obtain best match between the simulated spectra and the experimental spectra in a training data set. The performance of the mathematical model and the associated optimized parameter set used in the CID spectra simulation was evaluated by generating predictions for a large number of known peptides, which were not included in the training data set. It was shown that the model is able to predict peptide CID spectra with reasonable accuracy in fragment ion intensities for both singly and doubly charged peptide parent ions up to 2000 u in mass. The optimized parameter set was evaluated to gain insight into the collision-induced peptide fragmentation process.     

Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 μg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).     

Facile identification of phosphorylation sites in peptides by radical directed dissociation

Identification of phosphorylation sites is of interest due to their importance in protein regulation; however, the identification of the exact sites of this modification is not always easily obtained due to the dynamic nature of phosphorylation and the challenges faced during mass spectrometric analysis. Herein we elaborate on our previous communication (Diedrich, J. K.; Julian, R. R. J. Am. Chem. Soc. 2008, 130, 12212-12213) describing a novel technique for assignment of phosphorylation in a site-specific and facile manner. Phosphorylation sites are selectively modified through β elimination followed by Michael addition chemistry to install a photolabile group. Photodissociation with 266 nm light yields homolytic cleavage at the modification site, generating a β radical which is poised to fragment the peptide backbone. Dissociation primarily yields d-type ions at the previously phosphorylated residue, allowing facile identification. Radical directed fragmentation also occurs in smaller abundances at neighboring residues. The mechanisms behind this selective radical fragmentation are presented and the utility is discussed. Fragmentation is shown to be independent of charge state allowing analysis of a wide variety of peptide sequences including peptides with multiple phosphorylation sites. A comparison of this technique is made with collision induced dissociation (CID) and electron capture dissociation (ECD) for representative peptides.     

Low-energy collision-induced dissociation fragmentation analysis of cysteinyl-modified peptides

The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of Deinococcus radiodurans, the presence of these label-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys residue, and to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8.     

Computing fragmentation trees from tandem mass spectrometry data

The structural elucidation of organic compounds in complex biofluids and tissues remains a significant analytical challenge. For mass spectrometry, the manual interpretation of collision-induced dissociation (CID) mass spectra is cumbersome and requires expert knowledge, as the fragmentation mechanisms of ions formed from small molecules are not completely understood. The automated identification of compounds is generally limited to searching in spectral libraries. Here, we present a method for interpreting the CID spectra of the organic compound's protonated ions by computing fragmentation trees that establish not only the molecular formula of the compound and all fragment ions but also the dependencies between fragment ions. This is an important step toward the automated identification of unknowns from the CID spectra of compounds that are not in any database.     

Comprehensive phosphorylation site analysis of individual phosphoproteins applying scoring schemes for MS/MS data

We have developed novel scoring schemes for the identification of (phospho)peptides (PeptideScore) and for pinpointing phosphorylation sites (PhosphoSiteScore) using MS/MS data. These scoring schemes have been developed for the in-depth analysis of individual phosphoproteins, not for large-scale phosphoproteomic-type data. The scoring schemes are implemented into the new software tool Phosm, which provides a concise and comprehensive presentation of the results. For development and evaluation of these schemes, we have analyzed approximately 500 phosphopeptide MS/MS spectra, most of them nontryptic peptides. The novel scoring schemes turned out to be very powerful, even with CID MS/MS spectra of very low quality. Many phosphopeptides and phosphorylation sites that remained unassigned in our LC-MS/MS data sets with Mascot could be identified with Phosm. Especially the number of identified multiply phosphorylated peptides could be significantly increased. The applied scoring parameters are described, and the scoring for several selected examples of phosphopeptides is discussed in detail. Furthermore, a new and simple nomenclature for all types of phosphorylated fragment ions is introduced in this publication.     

PICquant: a quantitative platform to measure differential peptide abundance using dual-isotopic labeling with 12C6- and 13C6-phenyl isocyanate

We have developed a complete system for the isotopic labeling, fractionation, and automated quantification of differentially expressed peptides that significantly facilitates candidate biomarker discovery. We describe a new stable mass tagging reagent pair, (12)C(6)- and (13)C(6)-phenyl isocyanate (PIC), that offers significant advantages over currently available tags. Peptides are labeled predominantly at their amino termini and exhibit elution profiles that are independent of label isotope. Importantly, PIC-labeled peptides have unique neutral-mass losses upon CID fragmentation that enable charge state and label isotope identification and, thereby, decouple the sequence identification from the quantification of candidate biomarkers. To exploit these properties, we have coupled peptide fractionation protocols with a Thermo LTQ-XL LC-MS(2) data acquisition strategy and a suite of automated spectrum analysis software that identifies quantitative differences between labeled samples. This approach, dubbed the PICquant platform, is independent of protein sequence identification and excludes unlabeled peptides that otherwise confound biomarker discovery. Application of the PICquant platform to a set of complex clinical samples showed that the system allows rapid identification of peptides that are differentially expressed between control and patient groups.     

Complications in the assignment of 14 and 28 Da mass shift detected by mass spectrometry as in vivo methylation from endogenous proteins

Identification of protein methylation sites typically starts with database searching of MS/MS spectra of proteolytic digest of the target protein by allowing addition of 14 and 28 Da in the selected amino acid residues that can be methylated. Despite the progress in our understanding of lysine and arginine methylation, substrates and functions of protein methylation at other amino acid residues remain unknown. Here we report the analysis of protein methylation for p53, SMC3, iNOS, and MeCP2. We found that a large number of peptides can be modified on the lysine, arginine, histidine, and glutamic acid residues with a mass increase of 14 or 28 Da, consistent with methylation. Surprisingly, a majority of which did not demonstrate a corresponding mass shift when cells were cultured with isotope-labeled methionine, a precursor for the synthesis of S-adenosyl-l-methionine (SAM), which is the most commonly used methyl donor for protein methylation. These results suggest the possibility of either exogenous protein methylation during sample handling and processing for mass spectrometry or the existence of SAM-independent pathways for protein methylation. Our study found a high occurrence of protein methylation from SDS-PAGE isolated endogenous proteins and identified complications for assigning such modifications as in vivo methylation. This study provides a cautionary note for solely relying on mass shift for mass spectrometric identification of protein methylation and highlights the importance of in vivo isotope labeling as a necessary validation method.     

Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides

We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carried out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitin-conjugated proteins using LC/MS/MS.     

Approach for determining protein ubiquitination sites by MALDI-TOF mass spectrometry

Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Trypsin digestion of ubiquitin conjugated proteins produces diglycine branched peptides in which the C-terminal Gly-Gly fragment of ubiquitin is attached to the epsilon-amino group of a modified lysine residue within the peptide. This provides a platform for mapping ubiquitination sites using mass spectrometry. Here we report the development of a novel strategy for determining posttraslational protein ubiquitination based on the N-terminal sulfonation of diglycine branched peptides. In contrast to conventional tandem MS spectra of native tryptic peptides, MALDI MS/MS analysis of a sulfonated tryptic peptide containing a diglycine branch generates a unique spectrum composed of a signature portion and a sequence portion. The signature portion of the spectrum consists of several intense ions resulting from the elimination of the tags, the N-terminal residues at the peptide and the branch, and their combination. This unique ion distribution pattern can distinguish ubiquitination modificatons from others and can identify the first N-terminal residues of the peptides as well. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated diglycine branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. The technique is demonstrated for a series of synthetic peptides and is validated by a model protein, tetraubiquitin. Our results show that the MALDI MS/MS analysis of sulfonated tryptic peptides can provide a highly effective method for the determination of ubiquitination substrates, ubiquitination sites on protein targets, and modification sites on ubiquitins themselves.     

Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS

An automated screening method is presented that uses MALDI in-source decay (MALDI-ISD) of disulfide bonds for identification of disulfide-linked peptides in MALDI mass spectra. Peptides released by ISD of a disulfide bond can be detected at an m/z ratio that corresponds to the singly protonated peptide with a reduced cysteine residue. Therefore, screening of peak lists for signal patterns that fulfill the equation, m/z (peak A) + m/z (peak B) - m/z (H2 + H+) = m/z (peak C), facilitated identification of putative ISD fragments of disulfide-linked peptides (peaks A and B) and their precursors (peak C). Signals (peak C) from putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the existence of a disulfide bond. Using this method, we identified all 4 disulfide bonds in RNAseA and 8 two-disulfide clusters comprising 16 out of the 17 disulfide bonds in BSA. The presented screening method accelerated the identification of disulfide bonds in RNAseA and BSA, because the number of MS/MS spectra to be acquired was reduced by 1 order of magnitude. Less than 5% of the signals selected for LIFT-TOF/TOF-MS did not correspond to disulfide-linked peptides. Furthermore, the number of possible assignments for disulfide-linked peptides was reduced by 2-3 orders of magnitude, because knowledge of the mechanism of disulfide bond fragmentation by ISD permitted use of stricter rules for the interpretation of mass spectra. Therefore, interpretation of MS/ MS spectra of disulfide-linked peptides was considerably simplified in comparison to conventional approaches.     

Acid-labile isotope-coded extractants: a class of reagents for quantitative mass spectrometric analysis of complex protein mixtures

Quantitative mass spectrometry using stable isotope-labeled tagging reagents such as isotope-coded affinity tags has emerged as a powerful tool for identification and relative quantitation of proteins in current proteomic studies. Here we describe an integrated approach using both automated two-dimensional liquid chromatography/ mass spectrometry (2D-LC/MS) and a novel class of chemically modified resins, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. ALICE contains a thiol-reactive group that is used to capture all cysteine (Cys)-containing peptides from peptide mixtures, an acid-labile linker, and a nonbiological polymer. The acid-labile linker is synthesized in both heavy and light isotope-coded forms and therefore enables the direct relative quantitation of peptides/proteins through mass spectrometric analysis. To test the ALICE method for quantitative protein analysis, two model protein mixtures were fully reduced, alkylated, and digested in solution separately and then Cys-containing peptides covalently captured by either light or heavy ALICE. The reacted light and heavy ALICE were mixed and washed extensively under rigorous conditions and the Cys-containing peptides retrieved by mild acid-catalyzed elution. Finally, the eluted peptides were directly subjected to automated 2D-LC/MS for protein identification and LC/MS for accurate relative quantitation. Our initial study showed that quantitation of protein mixtures using ALICE was accurate. In addition, isolation of Cys-containing peptides by the ALICE method was robust and specific and thus yielded very low background in mass spectrometric studies. Overall, the use of ALICE provides improved dynamic range and sensitivity for quantitative mass spectrometric analysis of peptide or protein mixtures.     

Statistical characterization of ion trap tandem mass spectra from doubly charged tryptic peptides

Collision-induced dissociation (CID) is a common ion activation technique used to energize mass-selected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra.