title: A simplified method for peptide de novo sequencing using (18)O labeling.
abstract: Incorporation of an (18)O atom into a peptide C-terminus by proteolytic cleavage in the presence of H2(18)O is one of the most effective ways of enhancing tandem mass spectrometry (MS/MS)-based de novo sequencing. Incorporation is usually accomplished by procedures including vacuum-assisted drying of tryptic peptides extracted from gels, their subsequent reconstitution in a H2(16)O/H2(18)O mixture and re-treatment with trypsin. In the present work, we propose a simplified procedure for (18)O incorporation into tryptic peptides by adding H2(18)O and trypsin to the original digest solution. In comparison to published methods, the proposed protocol for peptide de novo sequencing brings significant advantages in analysis and workflow with no deterioration in method performance. We show that labeling by this simplified method leads to a highlighting of the y-ion fragment series in the peptide matrix-assisted laser desorption/ionization (MALDI)- MS/MS data, which facilitates MS/MS data interpretation. We also prove that eliminating acid extraction of peptides from gels does not result in a decrease in sequence coverage or a qualitative loss of particular peptides detectable by MALDI-MS. The method was examined by MALDI-MS/MS on bovine serum albumin and recombinant histidine kinase CKI1 from Arabidopsis thaliana, and was verified by de novo sequencing of tryptic peptides originating from Apodemus sylvaticus salivary proteins.
authors.affiliation: Research Group Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Aleš
authors.initials: A
authors.lastname: Voráĉ
authors.affiliation: Research Group Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Sedo
authors.affiliation: Research Group Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Havliš
authors.affiliation: Research Group Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic. zdrahal@sci.muni.cz.
authors.forename: Zbyněk
authors.initials: Z
authors.lastname: Zdráhal
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000032:analysis / Q000737:chemistry*; D008745:Methylation; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D010103:Oxygen Isotopes; D011494:Protein Kinases; C101990:CKI1 protein, Arabidopsis; D014357:Trypsin