Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry.
We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000-160,000 peaks above the predetermined threshold detectable in 60-microg cell protein samples), reproducibility (average coefficient of variance of 0.35-0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10(3)) of our platform warrant its application to various types of experimental and translational proteomics.
Ono
Masaya
M
Chemotherapy Division and Cancer Proteomics Project, National Cancer Center Research Institute, Tokyo 104-0045, Japan.
Shitashige
Miki
M
Honda
Kazufumi
K
Isobe
Tomohiro
T
Kuwabara
Hideya
H
Matsuzuki
Hirotaka
H
Hirohashi
Setsuo
S
Yamada
Tesshi
T
D000185:Actinin / Q000032:analysis / Q000378:metabolism; D001331:Automation / Q000295:instrumentation; D001773:Blood Cells / Q000737:chemistry; D002465:Cell Movement; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015179:Colorectal Neoplasms / Q000378:metabolism; D003952:Diagnostic Imaging; D004318:Doxycycline / Q000494:pharmacology; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D010190:Pancreatic Neoplasms / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012984:Software; D014162:Transfection; D014407:Tumor Cells, Cultured
C111453:ACTN4 protein, human; D008840:Microfilament Proteins; D010446:Peptide Fragments; D020543:Proteome; D000185:Actinin; D004318:Doxycycline