Precise peptide sequencing and protein quantification in the human proteome through in vivo lysine-specific mass tagging.
Proteomics studies demand new scalable and automatable MS-based methods with higher specificity and accuracy. Here we describe an accurate and efficient method for both precise quantification and comprehensive de novo identification of peptide sequences in complex mixtures. The unique feature of this method is based on the incorporation of deuterium-labeled (heavy) lysines into proteins through in vivo cell culturing, which introduces specific mass tags at the carboxyl termini of proteolytic peptides when cleaved by certain proteases. The mass shift between the unlabeled and the deuterated lysine (lys-d4) assigns a mass signature to all lysine-containing peptides in any pool of proteolytic peptides. Lys-d4 tags can also serve as internal markers in MS/MS fragment spectra when they are buried in some peptide sequences due to miscleavages. This signal specificity circumvents the mass accuracy limitations in determining particular amino acid residues for de novo sequencing. Further, this strategy of lysine-specific tagging was successfully implemented to measure the differential protein expression of human skin fibroblast cells in response to heat shock.
Gu
Sheng
S
Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Pan
Songqin
S
Bradbury
E Morton
EM
Chen
Xian
X
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D020543:Proteome / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
D010455:Peptides; D020543:Proteome; D014357:Trypsin; D008239:Lysine