Top-down quantitation and characterization of SILAC-labeled proteins.
Stable isotope labeling by amino acids in cell culture (SILAC) has become a popular labeling strategy for peptide quantitation in proteomics experiments. If the SILAC technology could be extended to intact proteins, it would enable direct quantitation of their relative expression levels and of the degree of modification between different samples. Here we show through modeling and experiments that SILAC is suitable for intact protein quantitation and top-down characterization. When SILAC-labeling lysine and/or arginine, peaks of light and heavy SILAC-doublets do not interfere with peaks of different charge states at least between 10 and 200 kDa. Unlike chemical methods, SILAC ensures complete incorporation-all amino acids are labeled. The isotopic enrichment of commercially available SILAC amino acids of nominally 95% to 98% shifts the mass difference between light and heavy state but does not lead to appreciably broadened peaks. We expressed labeled and unlabeled Grb2, a 28 kDa signaling protein, and showed that the two forms can be quantified with an average standard deviation of 6%. We performed on-line top-down sequencing of both forms in a hybrid linear ion trap orbitrap instrument. The quantized mass offset between fragments provided information about the number of labeled residues in the fragments, thereby simplifying protein identification and characterization.
Waanders
Leonie F
LF
Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Martinsried, Germany.
Hanke
Stefan
S
Mann
Matthias
M
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000032:analysis / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D040901:Proteomics
D051380:GRB2 Adaptor Protein; D011506:Proteins