TY - JOUR
T1 - Assessment of resolution parameters for CID-based shotgun proteomic experiments on the LTQ-orbitrap mass spectrometer
AU - Kim, Min Sik
AU - Kandasamy, Kumaran
AU - Chaerkady, Raghothama
AU - Pandey, Akhilesh
N1 - Funding Information:
The authors acknowledge support in part for this study by a grant S10RR023025 from the High End Instrumentation Program of the National Institutes of Health , a Department of Defense Era of Hope Scholar award ( W81XWH-06-1-0428 ), an NIH roadmap grant for Technology Centers of Networks and Pathways ( U54RR020839 ), and a contract N01-HV-28,180 from the National Heart Lung and Blood Institute .
PY - 2010/9
Y1 - 2010/9
N2 - Shotgun proteomics has been used extensively for characterization of a number of proteomes. High-resolution Fourier transform mass spectrometry (FTMS) has emerged as a powerful tool owing to its high mass accuracy and resolving power. One of its major limitations, however, is that the confidence level of peptide identification and sensitivity cannot be maximized simultaneously. Although it is generally assumed that higher resolution is better for peptide identifications, the precise effect of varying resolution as a parameter on peptide identification has not yet been systematically evaluated. We used the Escherichia coli proteome and a standard 48 protein mix to study the effect of different resolution parameters on peptide identifications in the setting of a shotgun proteomics experiment on an LTQ-Orbitrap mass spectrometer. We observed a higher number of peptide-spectrum matches (PSMs) whenever the MS scan was carried out by FT and the MS/MS in the ion-trap (IT) with the maximum PSMs obtained at an MS resolution of 30,000. In contrast, when samples were analyzed by FT for both MS and MS/MS, the number of PSMs was significantly lower (~40% compared with FT-IT experiments) with the maximum PSMs obtained when both the MS and MS/MS resolution were set to 15,000. Thus, a 15K-15K resolution setting may provide the best compromise for studies where both speed and accuracy such as high-throughput post-translational analysis and de novo sequencing are important. We hope that our study will allow researchers to choose between different resolution parameters to achieve their desired results from proteomic analyses.
AB - Shotgun proteomics has been used extensively for characterization of a number of proteomes. High-resolution Fourier transform mass spectrometry (FTMS) has emerged as a powerful tool owing to its high mass accuracy and resolving power. One of its major limitations, however, is that the confidence level of peptide identification and sensitivity cannot be maximized simultaneously. Although it is generally assumed that higher resolution is better for peptide identifications, the precise effect of varying resolution as a parameter on peptide identification has not yet been systematically evaluated. We used the Escherichia coli proteome and a standard 48 protein mix to study the effect of different resolution parameters on peptide identifications in the setting of a shotgun proteomics experiment on an LTQ-Orbitrap mass spectrometer. We observed a higher number of peptide-spectrum matches (PSMs) whenever the MS scan was carried out by FT and the MS/MS in the ion-trap (IT) with the maximum PSMs obtained at an MS resolution of 30,000. In contrast, when samples were analyzed by FT for both MS and MS/MS, the number of PSMs was significantly lower (~40% compared with FT-IT experiments) with the maximum PSMs obtained when both the MS and MS/MS resolution were set to 15,000. Thus, a 15K-15K resolution setting may provide the best compromise for studies where both speed and accuracy such as high-throughput post-translational analysis and de novo sequencing are important. We hope that our study will allow researchers to choose between different resolution parameters to achieve their desired results from proteomic analyses.
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U2 - 10.1016/j.jasms.2010.04.011
DO - 10.1016/j.jasms.2010.04.011
M3 - Article
C2 - 20483638
AN - SCOPUS:77955918485
SN - 1044-0305
VL - 21
SP - 1606
EP - 1611
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
IS - 9
ER -