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/microL), and ZipTipC(18) 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.
Pereira-Medrano
Ana Gabriela
AG
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Sheffield, United Kingdom.
Sterling
Alistair
A
Snijders
Ambrosius P L
AP
Reardon
Kenneth F
KF
Wright
Phillip C
PC
D000465:Algorithms; D045424:Complex Mixtures / Q000032:analysis / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D045424:Complex Mixtures; D011506:Proteins
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