Development and evaluation of Nano-ESI coupled to a triple quadrupole mass spectrometer for quantitative proteomics research

Digested BSA (Bovine Serum Albumin) was analyzed to optimize the conditions of the LC and the triple Q MS parameters.
BSA was first treated to break disulfide bonds with dithiothreitol and the cleaved bonds were then capped by 2-iodoacetamide. It was digested by Lys-C for 4h and digested by trypsin overnight. The digested BSA was desalinated by a SDB-XC filter. The conventional LC used ODS columns. Nano LC used ReprosilR particles packed into the fused silica nano spray tip. Digested BSA was separated on each column.
Most peptides in digested BSA were detected as protonated divalent or trivalent molecules ([M+2H] 2+ or [M+3H] 3+) in ESI positive ion mode. Monitoring the precursor ions measurement by Q1 SIM resulted in 81.5% coverage for the entire sequence of BSA. Mass spectrometric parameters for MRM analysis, such as product ion and collision energy, were optimized by an automatic MS optimization procedure.
The chromatographic and automatic MS optimization produced 97 MRM transitions for 10 peptides (limited to no more than 7 amino acids) for the nano-flow LC-MS method. Calibration curves were generated from each MRM transition with linearity improving when analyzed by nano-flow LC-MS as opposed to conventional LC-MS. Additionally, the LOD and LOQ of each transition analyzed by the nano-flow LC-MS method is several fold lower than the conventional LC-MS method. In summary, we improved the sensitivity of a triple Q MS by reducing the LC flow rate to nL/min. A few femtomoles of injected peptides could be analyzed by nano-flow LC-MS.
In this study, we developed and evaluated a novel quantitative system for proteomics with the result suggesting that this system could be applicable as a tool for shotgun proteomics.