Protein quantification by MALDI-selected reaction monitoring mass spectrometry using sulfonate derivatized peptides.
The feasibility of protein absolute quantification with matrix-assisted laser desorption/ionization (MALDI) using the selected reaction monitoring (SRM) acquisition mode on a triple quadrupole linear ion trap mass spectrometer (QqQ(LIT)) equipped with a high-frequency laser is demonstrated. A therapeutic human monoclonal antibody (mAb) was used as a model protein, and four tryptic peptides generated by fast tryptic digestion were selected as quantification surrogates. MALDI produces mostly singly charged peptides which hardly fragment under low-energy collision-induced dissociation (CID), and therefore the benefits of using 4-sulfophenyl isothiocyanate (SPITC) as a fragmentation enhancer derivatization agent were evaluated. Despite a moderate impact on the sensitivity, the N-terminus sulfonated peptides generate nearly complete y-ion ladders when native peptides produce few fragments. This aspect provides an alternative SRM transition set for each peptide. As a consequence, SRM transitions selectivity can be tuned more easily for peptide quantitation in complex matrices when monitoring several SRM transitions. From a quantitative point of view, the signal response depending on mAb concentration was found to be linear over 2.5 orders of magnitude for the most sensitive peptide, allowing precise and accurate measurement by MALDI-SRM/MS.
Lesur
Antoine
A
Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.
Varesio
Emmanuel
E
Hopfgartner
Gérard
G
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001557:Benzenesulfonates / Q000737:chemistry; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001557:Benzenesulfonates; D017879:Isothiocyanates; D010455:Peptides; D011506:Proteins; C015308:4-isothiocyanatobenzenesulfonate; D014357:Trypsin