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13 C‐Phenylisocyanate‐ A stable‐isotope mass tag for quantitative MS biomarker discovery in complex protein mixtures
Author(s) -
Templeton Dennis,
Lyons Charles,
Moshnikov Sergey,
Van Sant Christina,
Atkins Kristin,
Brenin David,
Cross Janet V.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a64-d
Subject(s) - chemistry , peptide , chromatography , reagent , mass spectrometry , phenylisocyanate , biomarker , isotope , biochemistry , physics , organic chemistry , quantum mechanics
Quantification of potential protein markers of disease using LC/MS is particularly difficult because of irreproducibility during chromatography and MS analyses. This can potentially be overcome with the use of internal standards, such as through the use of covalent stable‐isotope tagging methods. We have examined a new peptide labeling reagent, 13 C phenylisocyanate (PIC), which specifically labels the amino termini of peptides, and increases the mass of the labeled peptide 6 Da more than a paired peptide labeled with 12 C PIC. This reagent has several advantages over other mass tagging reagents currently in use: Peptides labeled with either 12 C and 13 C PIC precisely co‐elute in LC, enabling direct quantitation of spectral intensity using high precision MS1 ‘zoom’ scans such as in the figure below, which we have adapted to semi‐automated quantitation of ion pair intensities. In addition, characteristic neutral loss properties of PIC‐labeled peptides can distinguish genuine PIC‐labeled singlet marker peptides from singlets resulting from unlabeled peptides. Lastly, the PIC label can also identify b‐ions in MS2 spectra to enhance peptide sequencing.We are presently using this new mass tag label towards identifying proteins present in ductal lavage specimens that evidence human breast cancer, and will present our progress towards biomarker discovery using these complex protein mixtures.