Charge derivatization of peptides for analysis by mass spectrometry

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: Matrix-assisted Laser Desorption/ionization Tof/ Tof Mass Spmentioning

confidence: 99%

“…Attempts to simplify and improve the fragmentation pattern, both for PSD and MS/MS approaches, have largely concentrated on the introduction of charged groups at the N-or C-terminus of the peptide (for a review, see [10]). For these purposes chemical derivatizations that add acidic [11,12], fixed-charge [13], or basic [14] moieties have been used.…”

mentioning

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

“…Chemical modification reactions (also known as derivatizations) at the basic sites of peptides have been used to improve the fragmentation process [9]. The preformed charge of the derivative seems to count for an important role in the dissociation behavior of peptides [10]. The aim of many derivatization attempts was to place additional charges on the peptide [11,12,13].…”

mentioning

confidence: 99%

“…Oxidation of free cysteine to cysteic acid introduced a negative charge on the peptide and changed the dissociation behavior dramatically. As a consequence, a completely different approach has been carried out by Roth et al who derivatized peptides with a negative charge [10,22] to improve the fragmentation of singly charged peptides in MALDI-PSD spectra. The introduction of a negative charge (e.g., sulfonic acid) is counterbalanced by a second mobile proton.…”

mentioning

confidence: 99%

Investigation of the influence of charge derivatization on the fragmentation of multiply protonated peptides

Sonsmann¹,

Römer²,

Schomburg³

2002

The fragmentation of the multiply charged peptides b-chain of bovine insulin and glucagon have been investigated under low energy collision induced dissociation (CID) conditions using an electrospray ion trap mass spectrometer. The influence of charge state, specific amino acids such as aspartate or proline, the location of basic sites, and the derivatization on the fragmentation behavior has been the focus of interest. As a basis for understanding the fragmentation process, the concept of the mobile proton was applied. A set of different derivatives was used to manipulate the sites of protonation of the peptides in order to control and improve the fragmentation behavior. These results can be applied for de novo sequencing, although the sequence-specific fragmentation processes have significant influence on the dissociation behavior of the peptides. (J Am Soc Mass Spectrom 2002, 13, 47-58)

“…Multiple charging in electrospray can be enhanced by chemical derivatization to introduce fixed-charge or readily chargeable groups [3]. Several charge tags have been investigated on the basis of their chemical availability or synthetic convenience, e.g., 2,4,6-trimethylpyridinium [4], 2,2'-bipyridyl [5], tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) [6][7][8], and trimethylammoniumalkyl [9,10].…”

mentioning

confidence: 99%

Tunable Charge Tags for Electron-Based Methods of Peptide Sequencing: Design and Applications

Zimnicka

Moss

Chung

et al. 2011

Charge tags using basic auxiliary functional groups 6-aminoquinolinylcarboxamido, 4-aminopyrimidyl-1-methylcarboxamido, 2-aminobenzoimidazolyl-1-methylcarboxamido, and the fixedcharge 4-(dimethylamino)pyridyl-1-carboxamido moiety are evaluated as to their properties in electron transfer dissociation mass spectra of arginine C-terminated peptides. The neutral tags have proton affinities that are competitive with those of amino acid residues in peptides. Charge reduction by electron transfer from fluoranthene anion-radicals results in peptide backbone dissociations that improve sequence coverage by providing extensive series of N-terminal ctype fragments without impeding the formation of C-terminal z fragments. Comparison of ETD mass spectra of free and tagged peptides allows one to resolve ambiguities in fragment ion assignment through mass shifts of c ions. Simple chemical procedures are reported for Nterminal tagging of Arg-containing tryptic peptides.