Sequence dependent fragmentation of peptides generated by MALDI quadrupole time-of-flight (MALDI Q-TOF) mass spectrometry and its implications for protein identification

175

127

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/ MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques. (J Am Soc Mass Spectrom 2003, 14, 971-979)

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confidence: 93%

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confidence: 99%

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

Bodnar

Blackburn

Krise

et al. 2003

175

127

“…Statistical [19 -24] and mechanistic [25][26][27][28][29][30][31][32] analyses have led to a better understanding of peptide fragmentation behavior. Specifically, these studies have revealed residuespecific preferential cleavage N-terminal to proline (Pro), C-terminal to aspartic acid (Asp) and glutamic acid (Glu), and C-terminal to oxidized cysteine (e.g., cysteine sulfinic acid and cysteine sulfonic acid) [19,20,[33][34][35][36][37][38][39][40][41][42][43]. The dominance of these preferential cleavages may lead to a loss of fragmentation information because other fragment peaks may be of very low intensity or not present.…”

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confidence: 99%

Computational investigation and hydrogen/deuterium exchange of the fixed charge derivative tris(2,4,6-Trimethoxyphenyl) phosphonium: Implications for the aspartic acid cleavage mechanism

Herrmann

Wysocki

Vorpagel

2005

Aspartic acid (Asp)-containing peptides with the fixed charge derivative tris(2,4,6-trimethoxyphenyl) phosphonium (tTMP-P ϩ ) were explored computationally and experimentally by hydrogen/deuterium (H/D) exchange and by fragmentation studies to probe the phenomenon of selective cleavage C-terminal to Asp in the absence of a "mobile" proton. Ab initio modeling of the tTMP-P ϩ electrostatic potential shows that the positive charge is distributed on the phosphonium group and therefore is not initiating or directing fragmentation as would a "mobile" proton. Geometry optimizations and vibrational analyses of different Asp conformations show that the Asp structure with a hydrogen bond between the side-chain hydroxy and backbone carbonyl lies 2.8 kcal/mol above the lowest energy conformer. In reactions with D 2 O, the phosphonium-derived doubly charged peptide (H ϩ )P ϩ LDIFSDF rapidly exchanges all 12 of its exchangeable hydrogens for deuterium and also displays a nonexchanging population. With no added proton, P ϩ LDIFSDF exchanges a maximum of 4 of 11 exchangeable hydrogens for deuterium. No exchange is observed when all acidic groups are converted to the corresponding methyl esters. Together, these H/D exchange results indicate that the acidic hydrogens are "mobile locally" because they are able to participate in exchange even in the absence of an added proton. Fragmentation of two distinct (H ϩ )P ϩ LDIFSDF ion populations shows that the nonexchanging population displays selective cleavage, whereas the exchanging population fragments more evenly across the peptide backbone. This result indicates that H/D exchange can sometimes distinguish between and provide a means of separation of different protonation motifs and that these protonation motifs can have an effect on the fragmentation. (J Am Soc Mass Spectrom 2005, 16, 1067-1080

“…The completeness of the observed fragmentation series depends on the particular sequence of the peptide under study, the charge state of the ion produced, and the mode by which the fragmentation is induced within the mass spectrometer. By and large, doubly charged peptide ions tend to fragment more evenly across a given sequence than do singly charged ion species ( [40,44,51] and extensive personal observations). Thus, a large proportion of de novo sequencing has been performed on doubly charged ions, which are readily produced from tryptic peptides by electrospray ionization (ESI).…”

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confidence: 99%

“…By contrast, MALDI produces predominantly singly charged ions from peptides. These singly charged ion species often yield relatively low complexity CID mass spectra that tend to be dominated by a few preferred fragmentation pathways [40,[52][53][54][55][56]. When this fragmentation is induced on the slow time scale of resonant excitation within an ion trap mass spectrometer, the dissociation of singly-charged ions becomes especially selective, occurring largely adjacent to any aspartyl, glutamyl, or prolyl residues that may be present in the peptide [54,56].…”

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confidence: 99%

“De novo” peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: A preliminary study

Zhang

Krutchinsky

Chait

2003

Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal. Chem. 2001, 73, 5066 -5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-tonoise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process. T he use of mass spectrometry combined with database searching has gained wide acceptance for rapid, sensitive protein identification [1]. This method requires the availability of extensive protein, cDNA, or genomic sequence from the organism of interest (or at least from a closely related species). If such sequence information is not available, as remains the case for the vast majority of extant organisms, it is desirable to utilize de novo peptide sequencing to identify and obtain information about their protein(s). Although Edman sequencing has for a long time been a method of choice for de novo sequencing of peptides and proteins, mass spectrometry (MS) has also been shown to have considerable utility for this purpose.Indeed, mass spectrometric de novo sequencing based on the fragmentation of derivitized peptide ions dates back to the late 1960s and on underivatized peptide ions to the early 1980s (reviewed in [2] and [3]). More recently, MS sequencing has been given considerable impetus by the development of ESI and MALDI, ionization techniques whose efficiencies for producing intact peptide ions are far higher than those that were previously available. Again, both derivitized and underivatized peptides have been used in this newer work. Thus, chemical derivatization has been applied to simplify/enhance the fragmentation spectra and/or to differentiate N-from C-terminal fragment ions [4 -13]. Notable in this regard is the use of 18 O i...