Yield of 6,000 proteins by 1D nLC-MS/MS without pre-fractionation.
Mass spectrometry (MS) has dominated over other protein analysis methods that aspire to deliver rapid and sensitive protein annotation, due to its ability to acquire high-content biological information from samples of great complexity. Routinely, in-depth analysis of complex biological samples, such as total cell lysates, relies on the high separation power of two-dimensional liquid chromatography-tandem MS (2D LC-MS/MS), often combined with protein pre-fractionation. However, on the basis of recent advances in chromatographic and MS instrumentation, one-dimensional (1D) LC-MS/MS approaches have become the method-of-choice for high-volume/high-throughput protein experiments. Thousands of proteins can be identified in single-run LC-MS/MS experiments. In the present study a 1D LC-MS/MS approach was applied on whole-cell lysates of WM-266-4 human cells leading to identification of more than 5,300 protein groups, 6,000 proteins and 22,00 peptides, in a single run. Using no pre-fractionation steps, method optimization was achieved through experimentation on lysis and protein extraction solutions, as well as nLC gradient parameters.
Department of Proteomics, Division of Biotechnology, Center of Basic Research II, Biomedical Research Foundation of the Academy of Athens, Athens, Greece. Electronic address: atanagnost@bioacademy.gr.
Athanasios K
AK
Anagnostopoulos
Department of Cell Biology and Biophysics, Faculty of Biology, School of Sciences, National and Kapodistrian University of Athens, Athens, Greece.
Dimitrios J
DJ
Stravopodis
Department of Proteomics, Division of Biotechnology, Center of Basic Research II, Biomedical Research Foundation of the Academy of Athens, Athens, Greece. Electronic address: gthtsangaris@bioacademy.gr.
George Th
GT
Tsangaris
D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D006801:Humans; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000191:economics / Q000295:instrumentation* / Q000379:methods
D010455:Peptides; D020543:Proteome
1D–LC–MS/MS; Liquid chromatography; Mass spectrometry; Method development; Orbitrap; Protein identification; Single-run analysis; nLC–MS/MS