Improved detection specificity for plasma proteins by targeting cysteine-containing peptides with photo-SRM

Abstract

Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as an alternative to immunoassays for protein quantification owing to faster development time and higher multiplexing capability. However, the SRM strategy is faced with the high complexity of peptide mixtures after trypsin digestion of whole plasma or the cellular proteome that most of the time causes contamination, irremediably, by interfering compounds in the transition channels monitored. This problem becomes increasingly acute when the targeted protein is present at a low concentration. In this work, the merit of laser-induced photo-dissociation in the visible region at 473 nm implemented in an hybrid quadrupole linear ion-trap mass spectrometer (photo-SRM) was evaluated for detection specificity of cysteine-containing peptides in a group of plasma proteins after tagging with a dabcyl chromophore. Compared with conventional SRM, photo-SRM chromatograms have improved detection specificity for most of peptides monitored. Comparison of the signals obtained for the best proteotypic peptides in SRM mode and those recorded by photo-SRM of cysteine-containing peptides for the same proteins reveals either increased (up to 10-fold) or similar signal to photo-SRM detection. Finally, photo-SRM has extended response linearity across a calibration plot obtained by diluting human plasma in rat plasma, down to the lowest concentrations. Hence, photo-SRM may advantageously complement conventional SRM in assay of proteins in complex biological matrices.

Action-FRET: Probing the Molecular Conformation of Mass-Selected Gas-Phase Peptides with Förster Resonance Energy Transfer Detected by Acceptor-Specific Fragmentation

“…The acceptor chromophore is QSY7 Nsuccinimidyl ester (Life technologies), a dark quencher absorbing at 560 nm in solution and known to fragment efficiently at 532 nm in the gas phase. 38 The donor chromophore is 5-carboxyrhodamine 575 N-succinimidyl ester (rh575) (Life Technologies), absorbing at 550 nm and fluorescing at 575 nm in solution. Rh575 was also used as the donor chromophore by Talbot et al 18 , and both the fluorescence excitation and emission spectra along with photofragmentation mass spectra have been previously studied in detail.…”

Implementing visible 473 nm photodissociation in a Q-Exactive mass spectrometer: towards specific detection of cysteine-containing peptides

“…We previously illustrated the substantial reduction of interfering signals when targeting cysteine-containing peptides derivatizated with this chromophore by photo-SRM in whole plasma hydrolysate. 24 Such a specific dissociation mode should also considerably simplify data processing in the context of bottom-up proteomic experiments.…”

Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LC-MS/MS

“…Usually, tryptic peptides containing cysteine residues are avoided as they undergo iodoacetamidation. However, methods using signature peptide containing a cysteine residue have been reported after accounting for any mass change occurring prior to mass spectrometric detection [6,7]. Missed cleavages can result inconsistent production of the signature peptide in turn impacting quantitation.…”

“…Eight internal standardization approaches were compared with respect to accuracy and precision in work flows with and without digestion. Both analogue IS standard proteins (eel and human calcitonin), SIL-salmon calcitonin, SIL-salmon calcitonin signature peptide [1][2][3][4][5][6][7][8][9][10][11], and the cleavable SIL-salmon calcitonin peptide [1][2][3][4][5][6][7][8][9][10][11] were commercially obtained. 18 O-labeled form of the signature peptide was synthesized in-house by isotope exchange with18 O-labeled water.…”