Targeted comparative proteomics by liquid chromatography/matrix-assisted laser desorption/ionization triple-quadrupole mass spectrometry.
Here we report the first application of a matrix-assisted laser desorption/ionization (MALDI) triple-quadrupole mass spectrometer for targeted proteomics. Employing an amine-specific isotopic labelling approach, the technique was validated using five randomly selected bovine serum albumin peptides differentially labelled at known ratios. An indirect benefit of the isotopic labelling technique is a significant enhancement of the a1 ion in tandem mass (MS/MS) spectra of all peptides studied. Therefore, the a1 ion was selected as the fragment ion for multiple reaction monitoring (MRM) in all cases, eliminating tedious method development and optimization. Accurate quantification was achieved with an average relative standard deviation (RSD) of 5% (n = 5) and a detection limit of 14 amol. The technique was then applied to validate an important virulence biomarker of the fungal pathogen Candida albicans, which was not accurately quantified using global proteomics experiment employing two-dimensional liquid chromatography/electrospray ionization tandem mass spectrometry (2D-LC/ESI)-MS/MS. Using LC/MALDI-MRM analysis of five tryptic peptides, the protein PHR1 was found to be upregulated in the hyphal (pathogenic) form of C. albicans by a factor of 7.7 +/- 0.8.
Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford St., Halifax, Nova Scotia, Canada B3H 3Z1.
Jeremy E
JE
Melanson
Kenneth A
KA
Chisholm
Devanand M
DM
Pinto
D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002176:Candida albicans / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000032:analysis; D007553:Isotope Labeling / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D037521:Virulence Factors / Q000032:analysis
D015415:Biomarkers; D005656:Fungal Proteins; D010446:Peptide Fragments; D020543:Proteome; D037521:Virulence Factors; D012710:Serum Albumin, Bovine