A Systematic Evaluation of Chip-Based Nanoelectrospray Parameters for Rapid Identification of Proteins from a Complex Mixture

HPLC-MS/MS is widely used for protein identification from gel spots and shotgun fractions. Although HPLC has well recognized benefits, this type of sample infusion also has some undesirable attributes: relatively low sample throughput, potential sample-to-sample carryover, time-varying sample composition, and no option for longer sample infusion for longer MS analyses. An automated chip-based ESI device (CB-ESI) has the potential to overcome these limitations. This report describes a systematic evaluation of the information-dependant acquisition (IDA) and sample preparation protocols for rapid protein identification from a complex mixture using a CB-ESI source compared with HPLC-ESI (gradient and isocratic elutions). Cytochrome c and a six-protein mixture (11–117 kDa) were used to develop an IDA protocol for rapid protein identification and to evaluate the effects of sample preparation protocols. MS (1–10 s) and MS/MS (1–60 s) scan times, sample concentration (50–500 fmol/μL), and ZipTipC18 cleanup were evaluated. Based on MOWSE scores, protein coverage, experimental run time, number of identified proteins, and reproducibility, a 12.5 min experiment (22 cycles, each with one 3 s MS and eight 10 s MS/MS scans) was determined to be the optimal IDA protocol for CB-ESI. This work flow yielded up to 220% greater peptide coverage compared with gradient HPLC-ESI and provided protein identifications with up to a 2-fold higher throughput rate than either HPLC-ESI approach, whilst employing half the amount of sample over the same time frame. The results from this study support the use of CB-ESI as a rapid alternative to the identification of protein mixtures.