Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation

Abstract

We have demonstrated the use of per-methyl esterification of peptides for relative quantification of proteins between two mixtures of proteins and automated de novo sequence derivation on the same dataset. Protein mixtures for comparison were digested to peptides and resultant peptides methylated using either d0- or d3-methanol. Methyl esterification of peptides converted carboxylic acids, such as are present on the side chains of aspartic and glutamic acid as well as the carboxyl terminus, to their corresponding methyl esters. The separate d0- and d3-methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Parent proteins of methylated peptides were identified by correlative database searching of peptide tandem mass spectra. Ratios of proteins in the two original mixtures could be calculated by normalization of the area under the curve for identical charge states of d0- to d3-methylated peptides. An algorithm was developed that derived, without intervention, peptide sequence de novo by comparison of tandem mass spectra of d0- and d3-peptide methyl esters.

Expanding the organismal scope of proteomics: Cross‐species protein identification by mass spectrometry and its implications

“…One method to facilitate this interpretation is to enrich a series of fragments by attaching a strongly positively or strongly negatively charged group to the N-terminus of peptides [16,17]. Another method is to introduce an isotopic label to the C-terminus of peptides by digesting proteins in a buffer containing H 2 18 O (protocols reviewed in [6]) or by CD 3 OH [18]. 18 Olabeled y-ions can be recognized by a one or two Thomson shift (depending on the peptide's charge) and allow confident readout of the peptide sequence [19,20].…”

“…Another method is to introduce an isotopic label to the C-terminus of peptides by digesting proteins in a buffer containing H 2 18 O (protocols reviewed in [6]) or by CD 3 OH [18]. 18 Olabeled y-ions can be recognized by a one or two Thomson shift (depending on the peptide's charge) and allow confident readout of the peptide sequence [19,20]. The quality and the number of produced sequences enabled cloning of a few proteins via oligonucleotide primers and PCR.…”

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

“…Database searching can accommodate posttranslational modification of peptides by allowing specific, user-defined modifications [10,11]. If a complete ion series is present, the entire peptide sequence may be determined de novo without a database [12,13]. This approach is useful for proteins from unsequenced genomes.…”

“…Automated, high-throughput identification of proteins by MS/MS and database sequencing algorithms is successful for many samples [1,3,4,8,[12][13][14][15][16][17], but this approach sometimes fails to identify the protein because of inaccurate algorithm prediction [18]. Statistical [19 -24] and mechanistic [25][26][27][28][29][30][31][32] analyses have led to a better understanding of peptide fragmentation behavior.…”

Shifting unoccupied spectral space in mass spectrum of peptide fragment ions

“…While chemical modification of Tyr is not a common procedure in peptide mass spectrometry, esterification of peptidyl carboxylates using methanolic HCl is routinely used in phosphopeptide and phosphoproteome analysis [29]. This chemical reaction is generally considered to be quantitative, and reagents used can be readily removed.…”