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

Abstract

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...

Toward top-down determination of PEGylation site using MALDI in-source decay MS analysis

“…In this experiment, PEG being a large and heterogeneous molecule, MS sequencing information on Lys 19 is missing as a result of PEG attachments. As fragments for the modified side chains of proteins, such as phosphates, glycans, or disulfide bonds, are typically not observed in the spectra from ISD, ECD, and ETD, in contrast to CID [30], the truncation of the ion series at Lys 19 is in line with the prediction that a conjugation with a 20 kDa PEG would truncate the sequence readout at that particular position. It is believed that our observation resembles the case of disulfide bonds characterization or the study of proline containing sequences, where their presence is characterized by missing sequencing information due to the presence of cyclic moiety [32].…”

“…Due to its remarkable sensitivity, low sample consumption, ease of sample preparation, and high-throughput capability, MALDI-TOF MS has also been successfully used for the analysis of synthetic polymers [20 -23]. Further advancement has led to the development of MALDI-TOF/TOF MS [24 -26], which has been extensively used in proteomics applications for characterizing biomarkers and important post-translational modifications (PTMs) [27][28][29][30]. In addition, its reflector in-source decay (reISD) capability enabled the topdown analysis of intact proteins.…”

MALDI‐TOF/TOF de novo sequence analysis of 2‐D PAGE‐separated proteins from Halorhodospira halophila, a bacterium with unsequenced genome

“…It has been demonstrated that indirect evidence can add to the significance of an identification [20]. Therefore, the identifications were further validated by using information such as the cleavage specificity of trypsin and sequence information resulting from known preferential fragmentation patterns of sulfonated peptides [8].…”

“…Keough et al [6] demonstrated that the N-terminus can be derivatized by acylation with 2-sulfobenzoic acid cyclic anhydride or chlorosulfonylacetyl chloride. N-Terminal sulfonic acid derivatives were subsequently proposed for peptide sequencing by ESI-MS, MALDI-TOF MS [7], and MALDI-TOF/TOF MS [8]. The introduction of a sulfo group facilitates the MS/MS fragmentation of singly charged peptide ions by providing a second "mobile" proton which lowers amide bond strength and allows more facile unimolecular decay [9].…”

N-terminal tagging strategy forDe Novosequencing of short peptides by ESI-MS/MS and MALDI-MS/MS

“…We did not succeed in registering ESI spectrum of this peptide due to low stability of its molecular ion. Figure 4 clearly indicates that sulfobenzoylation results in increasing of y-series ion intensities as expected [40,46]. Head-to-tail cyclization is suppressed.…”