Peer Reviewed: Sulfonic Acid Derivatives for Peptide Sequencing by MALDI MS.

The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have increased its application in recent years. The most common method of "peptide mass fingerprint" analysis often does not provide robust identification. Additional sequence information, obtained by post-source decay or collision induced dissociation, provides additional constraints for database searches. However, de novo sequencing by mass spectrometry is not yet common practice, most likely because of the difficulties associated with the interpretation of high and low energy CID spectra. Success with this type of sequencing requires full sequence coverage and demands better quality spectra than those typically used for data base searching. In this report we show that full-length de novo sequencing is possible using MALDI TOF/TOF analysis. The interpretation of MS/MS data is facilitated by N-terminal sulfonation after protection of lysine side chains (Keough et al., Proc. Natl. Acad. Sci. U.S. A. 1999, 96, 7131-7136). Reliable de novo sequence analysis has been obtained using sub-picomol quantities of peptides and peptide sequences of up to 16 amino acid residues in length have been determined. The simple, predictable fragmentation pattern allows routine de novo interpretation, either manually or using software. Characterization of the complete primary structure of a peptide is often hindered because of differences in fragmentation efficiencies and in specific fragmentation patterns for different peptides. These differences are controlled by various structural parameters including the nature of the residues present. The influence of the presence of internal Pro, acidic and basic residues on the TOF/TOF fragmentation pattern will be discussed, both for underivatized and guanidinated/sulfonated peptides. (J Am Soc Mass Spectrom 2004, 15, 1838 -1852) © 2004 American Society for Mass Spectrometry P rotein identification protocols in proteomics currently rely on the peptide mass fingerprinting (PMF) technique in which a (mostly gel-purified) protein is digested with an endoprotease of known cleavage specificity. The masses of the resulting peptides are measured by mass spectrometry, usually matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), and matched to peptide masses that have been generated theoretically from proteins in databases. Notwithstanding the fact that the PMF approach is useful for identifying proteins in simple mixtures (e.g., 2D-PAGE gel spots), the identification of proteins in more complex mixtures often requires partial peptide sequence data obtained by tandem mass spectrometry (MS/MS) methods. If accurate genome sequence information is available, database search algorithms can be used in conjunction with MS/MS to readily identify proteins. For proteins not contained within sequence databases, it is necessary to determine partial or complete amino acid sequences using either manual or automated de novo peptide sequence analysis methods. This genera...

Section: De Novo Sequence Analysismentioning

confidence: 99%

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

Samyn

Debyser

Sergeant

et al. 2004

“…The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. M ass spectrometric characterization of disulfide bonded peptides is generally achieved by the reduction of the S-S bond followed by alkylation [1][2][3][4] or by oxidation to sulfonic acid derivatives [5]. Subsequent gas-phase fragmentation results in readily identifiable backbone cleavage products.…”

mentioning

confidence: 99%

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

Thakur

Balaram

2008

The fragmentation of positive and negative ions of peptide disulfides under mass spectrometric conditions yields distinctly different product ion distributions. A negative ion upon collision induced dissociation yields intense product ions, which correspond to cleavage at the disulfide linkage. The complete assignment of the product ions obtained upon fragmentation of oxidized glutathione in an ion trap is presented. The cleavage at the disulfide site is mediated by abstraction of C ␣ H and C ␤ p H protons resulting in product ions derived by neutral loss of H 2 S 2 and H 2 S. The formation of peptide thioaldehydes and persulfides at the cysteine sites is established. Dehydroalanine formation at the Cys residue is predominant. The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. M ass spectrometric characterization of disulfide bonded peptides is generally achieved by the reduction of the S-S bond followed by alkylation [1][2][3][4] or by oxidation to sulfonic acid derivatives [5]. Subsequent gas-phase fragmentation results in readily identifiable backbone cleavage products. Several groups have focused on the mass spectrometric analysis of peptides containing intact disulfide bonds using both positive and negative ions for gas-phase fragmentation [6 -12]. In the case of positively charged peptide ions, disulfide bond fragmentation occurs with much lower efficiency than cleavage of backbone peptide bonds, under conditions of collision induced dissociation with non-mobile protons suggested to play an important role [11]. Restricted proton mobility along the polypeptide backbone has also been implicated in cases of disulfide cleavages in polypeptides, which have been cationized using gold (I) [7]. The use of metal ions as gas-phase disulfide cleavage agents has also been investigated [12]. In general, only limited information regarding structure can be derived from intact disulfide peptides in the positive ion mode. In contrast, several recent studies have emphasized the utility of negative ion mass spectrometry [7,10,[13][14][15][16][17][18]. Specifically, Bowie and coworkers have presented assignments of product ion spectra obtained by negative ion electrospray mass spectrometry of peptides containing both intra and inter molecular disulfide bond [10,18]. The present study complements the work of Bowie and coworkers and further provides mechanistic details by examining second generation product ions by using ion trap mass spectrometry.In the structural analysis of peptides containing intact disulfides, under negative ion conditions, proton abstraction from the C ␣ H and C ␤ H positions of the Cys residue results in diverse fragmentation reactions, leading to cleavage of disulfide linkages. In the case of peptides that contain labile peptide bonds, for example X-Pro sequences, preferential cleavage at these sites results in two linear chains linked by a disulfide bond, which then undergo further f...

“…To this end, a number of different methods for controlling the fragmentation reactions of peptide ions, involving "fixed charge" derivatization, have been developed [25][26][27][28][29][30]. Until recently, most of this work has been directed toward derivatization of the N-and C-termini of the peptide of interest, or the side chains of lysine and arginine residues, and has largely been focused on directing fragmentation toward the formation of a series of "sequence" ions via cleavage along the amide backbone.…”

mentioning

confidence: 99%

Mechanisms for the selective gas-phase fragmentation reactions of methionine side chain fixed charge sulfonium ion containing peptides

Amunugama

Roberts

Reid

2006

To enable the development of improved tandem mass spectrometry based methods for selective proteome analysis, the mechanisms, product ion structures, and other factors influencing the gas-phase fragmentation reactions of methionine side-chain derivatized "fixed-charge" phenacylsulfonium ion containing peptide ions have been examined. Dissociation of these peptide ions results in the exclusive characteristic loss of the derivatized side chain, thereby enabling their selective identification. The resultant product ion(s) are then subjected to further dissociation to obtain sequence information for subsequent protein identification. Molecular orbital calculations (at the B3LYP/6-31 ϩ G (d,p) level of theory) performed on a simple peptide model, together with experimental evidence obtained by multistage dissociation of a regioselectively deuterated methionine derivatized sulfonium ion containing tryptic peptide, indicate that fragmentation of the fixed charge containing peptide ions occurs via S N 2 reactions involving the N-and C-terminal amide bonds adjacent to the methionine side chain, resulting in the formation of stable cyclic five-and six-membered iminohydrofuran and oxazine product ions, respectively. These studies further indicate that the rings formed via these neighboring group reactions are stable to further dissociation by MS 3 . As a consequence, the formation of b-or y-type sequence ions are "skipped" at the site of cyclization. Despite this, complete sequence information is still obtained because of the presence of both cyclic