MS/MS simplification by 355 nm ultraviolet photodissociation of chromophore-derivatized peptides in a quadrupole ion trap

Ultraviolet photodissociation (UVPD) of chromophore-modified peptides enhances the capabilities for de novo sequencing in a quadrupole ion trap mass spectrometer. Attachment of UV chromophores allows efficient photoactivation of not only the precursor ions but also any fragments that retain the chromophore functionality. For doubly protonated peptides, UVPD leads to a vast reduction in MS/MS complexity. The array of b and y ions typically seen upon collisionally activated dissociation is reduced to a single series of either y or b ions by UVPD depending on the location of the chromophore (i.e., N- or C-terminus). The sulfonation reagent Alexa Fluor 350 (AF350) provided the best overall results for the singly and doubly charged peptides by UVPD. The nonsulfonated analogue of AF350, 7-amino-4-methylcoumarin-3-acetic acid, also led to simplified spectra for doubly charged, but not singly charged, peptides by UVPD. Dinitrophenyl-peptides also yielded simplified spectra by UVPD albeit with a small amount of internal fragments accompanying the series of diagnostic y ions. The success of this MS/MS simplification process stems from extensive secondary fragmentation of any chromophore-containing fragments upon exposure to subsequent laser pulses. Energy-variable UVPD reveals that the abundances of non-chromophore-containing y fragment ions increase linearly with laser pulse energy, suggesting secondary dissociation of these species is insignificant. The abundances of chromophore-containing a/b fragment ions follow a quadratic trend due to the extensive secondary fragmentation at higher laser energies or multiple pulses.     

De novo sequencing of neuropeptides using reductive isotopic methylation and investigation of ESI QTOF MS/MS fragmentation pattern of neuropeptides with N-terminal dimethylation

A stable-isotope dimethyl labeling strategy was previously shown to be a useful tool for quantitative proteomics. More recently, N-terminal dimethyl labeling was also reported for peptide sequencing in combination with database searching. Here, we extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue extract without any genomic information. We demonstrated several new sequencing applications of this method in addition to the identification of the N-terminal residue using the enhanced a(1) ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y((n-1)) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also observed. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of N(alpha), N(epsilon)-tetramethylated lysine and N(epsilon)-dimethylated lysine are explored.     

Investigation of neutral loss during collision-induced dissociation of peptide ions

MS/MS fragmentation of peptides is dominated by overlapping b and y ion series. However, alternative fragmentation possibilities exist, including neutral loss. A database was generated containing 8400 MS/MS spectra of tryptic peptides assigned with high probability to an amino acid sequence (true positives) and a set of certified false (true negative) assignments for analysis of the amino terminus. A similar database was created for analysis of neutral loss at the carboxy termini using a data set of chymotryptic peptides. The analysis demonstrated that the presence of an internal basic residue, limiting proton mobility, has a profound effect on neutral loss. Peptides with fully mobile protons demonstrated minimal neutral loss, with the exception of amide bonds with proline on the carboxy terminal side, which created an intense neutral loss peak. In contrast, peptides with partial proton mobility contained many amino acids on either side of the amide bond associated with a strong neutral loss peak. Most notable among these was proline on the carboxy terminal side of an amide bond and aspartic acid on the amino terminal side of a bond. All results were found to be consistent for doubly and triply charged peptides and after adjustment for pairings across the amide bonds with particularly labile residues. The carboxy terminal of chymotryptic peptides also demonstrated significant neutral loss events associated with numerous amino acid residues. Clarification of the rules that govern neutral loss, when incorporated into analysis software, will improve our ability to correctly assign spectra to peptide sequences.     

Charge-remote metastable ion decomposition of free fatty acids under FAB MS: evidence for biradical ion structures

Classical charge-remote fragmentation (CRF) of a series of long-chain saturated and monounsaturated fatty acid anions, a well-known phenomenon under collisional activation conditions, is observed for the first time during fast atom bombardment of the analyte-matrix mixture without collisional activation. The process is efficient enough to allow collision-induced dissociation and metastable ion decomposition MS/MS spectra of any charge-remote [M-H2-(CH2)n]- fragments as well as spectra of neutral losses to be recorded. The results obtained are in contradiction to the generally accepted theory that CRF results exclusively in terminally unsaturated carboxylate anions. The new results indicate that a multistep radical mechanism is involved in CRF ion formation. The first step of the process appears to be accompanied by hydrogen elimination that occurs randomly throughout the molecule. The primary fragment radical ions formed can decompose further with the formation of the next generation of CRF ions.     

Screening and sequencing of glycated proteins by neutral loss scan LC/MS/MS method

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. A reversed-phase liquid chromatography method followed by a neutral loss scan mass spectrometric method was developed for the screening of glycation in proteins. The neutral loss scan was based on a unique sugar moiety neutral loss (-162 Da) that we observed in the fragmentation spectra of glycated peptides on Q-Tof type mass spectrometers. The collision energy was optimized for this neutral loss using a glycated synthetic peptide, and 20 eV was found to be the optimum collision energy. The neutral loss scan experiment was composed of two segments. In the first segment, the glycated peptides were identified based on the signature neutral loss of 162 Da when the collision energy was elevated to 20 eV. In the second segment, the glycated peptides were selected as the parent ions and fragmented at higher collision energy to break the peptide bonds. The fragmentation spectra of the selected glycated peptides revealed both the amino acid sequences and the sites of glycation. This neutral loss scan method was used to study the glycation in human serum albumin (HSA). The glycation sites in HSA were identified based on the retention time shift of glycated peptides, the mass accuracy from the MS scan, the signature neutral loss, and MS/MS information. Using this method, we were able to identify that 31 lysine residues were partially glycated from the glycated HSA sample, which has a total of 59 lysine residues.     

De novo peptide sequencing by two-dimensional fragment correlation mass spectrometry

A novel concept of two-dimensional fragment correlation mass spectrometry and its application to peptide sequencing is described. The daughter ion (MS2) spectrum of a peptide contains the sequence information of the peptide. However, deciphering the MS2 spectrum, and thus deriving the peptide sequence is complex because of the difficulty in distinguishing the N-terminal fragments (e.g., b series) from the C-terminal fragments (e.g., y series). By taking a granddaughter ion (MS3) spectrum of a particular daughter ion, all fragment ions of the opposite terminus are eliminated in the MS3 spectrum. However, some internal fragments of the peptide will appear in the MS3 spectrum. Because internal fragments are rarely present in the MS2 spectrum, the intersection (a spectrum containing peaks that are present in both spectra) of the MS2 and MS3 spectra should contain only fragments of the same terminal type. A two-dimensional plot of the MS2 spectrum versus the intersection spectra (2-D fragment correlation mass spectrum) often gives enough information to derive the complete sequence of a peptide. This paper describes this novel technique and its application in sequencing cytochrome c and apomyoglobin. For a tryptic digest of cytochrome c, approximately 78% of the protein sequence was determined. For the Glu-C/tryptic digest of apomyoglobin, approximately 66% of the protein sequence was determined.     

Using statistical models to identify factors that have a role in defining the abundance of ions produced by tandem MS

A database of 5448 peptide tandem mass spectra acquired in a quadrupole time-of-flight mass spectrometer was generated for peptides derived from proteins digested with trypsin. Peptides were identified from their mass spectra by the Mascot algorithm. Statistical models were then used to investigate factors influencing the abundance of ions formed. Separate models were formulated for b and y ions as it was thought that different factors may influence the formation of each type of ion. Several factors were found to have a highly significant influence on the abundance of ions formed. These include the actual mass of the ion formed after fragmentation as well as the location of the cleavage. The composition of the fragmenting peptide was also found to be important, and amino acids either side of the fragmentation site influenced the abundance of ions produced. To increase understanding of fragmentation mechanisms, the effect of several physicochemical properties of these residues was also investigated in a separate model. In conclusion, the models formulated for b and y ions provide useful characterization of the abundance of ions formed, and this information could be used to develop improved algorithms for peptide identification.     

Primary structure determination of peptides and enzymatically digested proteins using capillary liquid chromatography/mass spectrometry and rapid linked-scan techniques

Primary protein sequences were determined for both peptides and enzymatically digested proteins by rapid linked-scan (B/E) liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) at the low-picomole level (10-50 pmol). During the course of a single LC/MS/MS analysis, we demonstrated that it is possible to generate interpretable collision-induced dissociation spectra of the eluting proteolytic peptides. Molecular weights of tryptic peptides were established by using 1/10 of the protein digest by operating in the capillary LC/frit-FABMS mode. Peptides exhibiting the strongest MH+ ions were then selected for subsequent LC/MS/MS analysis (typically 1/5 of the remaining protein digest). Elution times for each chromatographic peak were generally greater than 30 s. It was therefore possible to obtain a minimum of six B/E fast linked-scan spectra during the course of elution of each peptide component. Typically, B/E linked scans of the greatest ion abundance (obtained at the chromatographic peak maximum) were averaged to enhance the signal/noise ratio at these low-picomole levels. Unit resolution was observed for product ions below m/z 1000. Rapid linked scanning by LC/frit-FABMS/MS provided mass assignments for product ions within 0.2-0.3 amu of theoretical values. Side-chain fragment ions (wn and dn) were also observed, which allowed for the differentiation of isobaric amino acids (e.g., leucine and isoleucine). Examples of the application of this fast linked-scan technique to LC/MS/MS are presented for complex mixtures of unknown peptides and the tryptic digestion of phosphorylated beta-casein.     

Submicrosecond surface-induced dissociation of peptide ions in a MALDI TOF MS

Surface-induced dissociation (SID) has been implemented in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI TOF MS), allowing production of tandem mass spectrometric information for peptide ions (MALDI TOF SID TOF). The instrument retains the standard operational modes such as the reflectron monitoring of the MALDI-generated intact ions and postsource decay. We show through ion trajectory simulations and experimental results that implementing SID in a commercial MALDI TOF spectrometer is feasible and that the SID products in this instrument fall in an observation time frame that allows the specific detection of fast-fragmentation channels. The instrument design, pulse timing sequence, and high-voltage electronics together with SID spectra of MALDI-generated peptide ions are presented. Standard peptides such as YGGFLR, angiotensin III, fibrinopeptide A, and des-Arg1-bradykinin were dissociated by means of hyperthermal collisions with a gold surface coated with a self-assembled monolayer of 2-(perfluorodecyl)ethanethiol. With the extraction fields and the short observation times used, the spectra obtained show intense low-mass ion signals such as immonium, b2, b3, and y2 ions. TOF data analysis involved matching simulated and experimental flight times and indicates that the observed fragments are produced at approximately 250 ns after the precursor ion collides with the surface. This submicrosecond gas-phase fragmentation time frame is complementary to the observation time frame of existing SID spectrometers, which are on the order of 10 micros for tandem quadrupoles and are larger than a few milliseconds for SID implemented in Fourier transform ion cyclotron resonance spectrometers.     

Automated iterative MS/MS acquisition: a tool for improving efficiency of protein identification using a LC-MALDI MS workflow

We have developed an information-dependent, iterative MS/MS acquisition (IMMA) tool for improving MS/MS efficiency, increasing proteome coverage, and shortening analysis time for high-throughput proteomics applications based on the LC-MALDI MS/MS platform. The underlying principle of IMMA is to limit MS/MS analyses to a subset of molecular ions that are likely to identify a maximum number of proteins. IMMA reduces redundancy of MS/MS analyses by excluding from the precursor ion peak lists proteotypic peptides derived from the already identified proteins and uses a retention time prediction algorithm to limit the degree of false exclusions. It also increases the utilization rate of MS/MS spectra by removing "low value" unidentifiable targets like nonpeptides and peptides carrying large loads of modifications, which are flagged by their "nonpeptide" excess-to-nominal mass ratios. For some samples, IMMA increases the number of identified proteins by ～20-40% when compared to the data dependent methods. IMMA terminates an MS/MS run at the operator-defined point when "costs" (e.g., time of analysis) start to overrun "benefits" (e.g., number of identified proteins), without prior knowledge of sample contents and complexity. To facilitate analysis of closely related samples, IMMA's inclusion list functionality is currently under development.     

Fragmentation characteristics of collision-induced dissociation in MALDI TOF/TOF mass spectrometry

The identification of proteins by tandem mass spectrometry relies on knowledge of the products produced by collision-induced dissociation of peptide ions. Most previous work has focused on fragmentation statistics for ion trap systems. We analyzed fragmentation in MALDI TOF/TOF mass spectrometry, collecting statistics using a curated set of 2459 MS/MS spectra and applying bootstrap resampling to assess confidence intervals. We calculated the frequency of 18 product ion types, the correlation between both mass and intensity with ion type, the dependence of amide bond breakage on the residues surrounding the cleavage site, and the dependence of product ion detection on residues not adjacent to the cleavage site. The most frequently observed were internal ions, followed by y ions. A strong correlation between ion type and the mass and intensity of its peak was observed, with b and y ions producing the most intense and highest mass peaks. The amino acids P, W, D, and R had a strong effect on amide bond cleavage when situated next to the breakage site, whereas residues including I, K, and H had a strong effect on product ion observation when located in the peptide but not adjacent to the cleavage site, a novel observation.     

193 nm ultraviolet photodissociation of imidazolinylated Lys-N peptides for de novo sequencing

The goal of many MS/MS de novo sequencing strategies is to generate a single product ion series that can be used to determine the precursor ion sequence. Most methods fall short of achieving such simplified spectra, and the presence of additional ion series impede peptide identification. The present study aims to solve the problem of confounding ion series by enhancing the formation of "golden" sets of a, b, and c ions for sequencing. Taking advantage of the characteristic mass differences between the golden ions allows N-terminal fragments to be readily identified while other ion series are excluded. By combining the use of Lys-N, an alternate protease, to produce peptides with lysine residues at each N-terminus with subsequent imidazolinylation of the ε-amino group of each lysine, peptides with highly basic sites localized at each N-terminus are generated. Subsequent MS/MS analysis by using 193 nm ultraviolet photodissociation (UVPD) results in enhanced formation of the diagnostic golden pairs and golden triplets that are ideal for de novo sequencing.     

Enabling MALDI-FTICR-MS/MS for high-performance proteomics through combination of infrared and collisional activation

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a central tool for proteomic analysis, yet the singly protonated tryptic peptide ions produced by MALDI are significantly more difficult to dissociate for tandem mass spectrometry (MS/MS) than the corresponding multiply protonated ions. In order to overcome this limitation, current proteomic approaches using MALDI-MS/MS involve high-energy collision-induced dissociation (CID). Unfortunately, the use of high-energy CID complicates product ion spectra with a significant proportion of irrelevant fragments while also reducing mass accuracy and mass resolution. In order to address the lack of a high-resolution, high mass accuracy MALDI-MS/MS platform for proteomics, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and a recently developed MS/MS technique termed CIRCA (for combination of infrared and collisional activation) have been applied to proteomic analysis. Here, CIRCA is shown to be suitable for dissociating singly protonated tryptic peptides, providing greater sequence coverage than either CID or infrared multiphoton dissociation (IRMPD) alone. Furthermore, the CIRCA fragmentation spectra are of sufficient quality to allow protein identification based on the MS/MS spectra alone or in concert with the peptide mass fingerprint (PMF). This is accomplished without compromising mass accuracy or mass resolution. As a result, CIRCA serves to enable MALDI-FTICR-MS/MS for high-performance proteomics experiments.     

Multiplexed MS/MS in a quadrupole ion trap mass spectrometer

A multiplexing method for performing MS/MS on multiple peptide ions simultaneously in a quadrupole ion trap mass spectrometer (QITMS) has been developed. This method takes advantage of the inherent mass bias associated with ion accumulation in the QITMS to encode the intensity of precursor ions in a way that allows the corresponding product ions to be identified. The intensity encoding scheme utilizes the Gaussian distributions that characterize the relationship between ion intensities and rf trapping voltages during ion accumulation. This straightforward approach uses only two arbitrary waveforms, one for isolation and one for dissociation, to gather product ion spectra from N precursor ions in as little as two product ion spectra. In the example used to illustrate this method, 66% of the product ions from five different precursor peptide ions were correctly correlated using the multiplexing approach. Of the remaining 34% of the product ions, only 6% were misidentified, while 28% of the product ions failed to be identified because either they had too low intensity or they had the same m/z ratio as one of the precursor ions or the same m/z ratio as a product ion from a different precursor ion. This method has the potential to increase sample throughput, reduce total analysis times, and increase signal-to-noise ratios as compared to conventional MS/MS methods.     

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.     

Improvement of the MS/MS fragment ion coverage of acidic residue-containing peptides by amidation with 15N-substituted amine

Tandem mass spectrometry (MS/MS) is a powerful tool for peptide sequencing and characterization. However, the selective cleavage at acidic residues, aspartic acid, and glutamic acid prevents the generation of enough product ions to elucidate the entire sequence. We attempted to solve the problem by converting the residues into the corresponding amides, asparagine and glutamine. The amidation suppressed the cleavage at the converted residues, and the product ions derived from dissociation at other sites became abundant. Incorporation of nitrogen isotope (15)N in the amine constituent for amidation minimized the mass change from -0.984 016 to +0.013 019, allowing easy discrimination of acidic and amide residues in the original sequences by MS/MS database search. In addition, the amidated and unchanged peptides had the same nominal mass, even when the transformation was incomplete, which was approximately 70% in the current condition. The unmodified acidic residues remaining were rather useful to give marker fragments by the dominant dissociation. These results demonstrate that (15)N-amidation is effective in improving the performance of MS/MS to elucidate amino acid sequences of peptides.     

Accurate mass-driven analysis for the characterization of protein phosphorylation. Study of the human Chk2 protein kinase

We describe the data-dependent analysis of protein phosphorylation using rapid-acquisition nano-LC-linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FTMS). The accurate m/z values of singly, doubly, and triply charged species calculated from the theoretical protonated masses of peptides phosphorylated at all Ser, Thr, or Tyr residues of the human checkpoint 2 (Chk2) protein kinase were used for selected ion extraction and chromatographic analysis. Using a kinase-inactive Chk2 mutant as a control, accurate mass measurements from FTMS and collision-induced dissociation spectra, 11 novel Chk2 autophosphorylation sites were assigned. Additionally, the presence of additional Chk2 phosphorylation sites in two unique peptides was deduced from accurate mass measurements. Selected ion chromatograms of all Chk2 phosphopeptides gave single peaks except in three cases in which two closely eluting species were observed. These pairs of phosphopeptides were determined to be positional isomers from MS/MS analysis. In this study, it was also found that ions due to the neutral loss of phosphoric acid from the parent peptide ion were not prominent in 18 of 36 MS/MS spectra of O-linked Chk2 phosphopeptides. Thus, accurate mass-driven analysis and rapid parallel MS/MS acquisition is a useful method for the discovery of new phosphorylation sites that is independent of the signature losses from phosphorylated amino acid residues.     

Utility of immonium ions for assignment of epsilon-N-acetyllysine-containing peptides by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) is a powerful tool for characterization of post-translationally modified proteins, including epsilon-N-acetyllysine-containing species. Previous reports indicate that epsilon-N-acetyllysine immonium ions are useful marker ions for peptides containing epsilon-N-acetyllysine, but the specificity and sensitivity of these ions for assignment of lysine acetylation by MS/MS have not been studied in detail. We investigated MS/MS data sets of 172 epsilon-N-acetyllysine tryptic peptides and 268 nonacetylated tryptic peptides to establish the utility and reliability of epsilon-N-acetyllysine immonium ions for identification and validation of acetylated peptides. Our analysis shows that the immonium ion at m/z 143 lacks specificity for lysine-acetylated peptides, whereas the derivative at m/z 126 is highly specific (98.1%). We also studied the positional effect of the epsilon-N-acetyllysine on the intensity of observed acetyllysine immonium ions. We observed an increase in acetyllysine immonium ion intensities when the acetylated lysine was N-terminally positioned in the peptide as compared to internal positions. Based on these observations we propose a validation scheme for unambiguous assignment of acetyllysine-containing peptides by MS/MS. Our analysis of epsilon-N-acetyllysine immonium ions provide a framework for investigation of MS/MS marker ion specificity and sensitivity that can be applied in studies of other types of post-translational modifications.     

Use of deuterium-labeled lysine for efficient protein identification and peptide de novo sequencing

Here, we describe a method for protein identification and de novo peptide sequencing. Through in vivo cell culturing, the deuterium-labeled lysine residue (Lys-d4) introduces a 4-Da mass tag at the carboxyl terminus of proteolytic peptides when cleaved by certain proteases. The 4-Da mass difference between the unlabeled and the deuterated lysine assigns a mass signature to all lysine-containing peptides in any pool of proteolytic peptides for protein identification directly through peptide mass mapping. Furthermore, it was used to distinguish between N- and C-terminal fragments for accurate assignments of daughter ions in tandem MS/MS spectra for sequence assignment. This technique simplifies the labeling scheme and the interpretation of the MS/MS spectra by assigning different series of fragment ions correctly and easily and is very useful in de novo peptide sequencing. We have also successfully implemented this approach to the analysis of protein mixtures derived from the human proteome.