TY - JOUR
T1 - One-dimensional capillary liquid chromatographic separation coupled with tandem mass spectrometry unveils the escherichia coli proteome on a microarray scale
AU - Iwasaki, Mio
AU - Miwa, Shohei
AU - Ikegami, Tohru
AU - Tomita, Masaru
AU - Tanaka, Nobuo
AU - Ishihama, Yasushi
PY - 2010/4/1
Y1 - 2010/4/1
N2 - We successfully identified the proteome expressed in Escherichia coli (E. coli) cells on a microarray scale using one-dimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a 350 cm long, 100 μm i. d., monolithic silica-C18 capillary column. E. coli tryptic digest (4 μg) was injected onto the column, and a 41 h gradient was applied with a flow rate of 500 nL/min at less than 20 MPa. In total, 22 196 nonredundant tryptic peptides from 2602 proteins, including 830 membrane proteins, were identified from the E. coli cells (triplicate analysis), in which an equivalent number of genes was detected by transcriptome analysis. Approximately a 5-fold larger peak response on average was obtained in this system, compared with that obtained by conventional capillary LC-MS/MS analysis with a 15 cm long, 3 μm diameter C18 silica particle-packed column. The higher response suggests that the influence of ionization suppression was drastically reduced by the high-efficiency separation on the long monolithic silica column coupled with the shallow gradient. Because this high-resolution system does not require any additional separation prior to LC-MS/MS, this "one-shot" proteomics approach can simplify the workflow of shotgun proteomics and minimize the sample amount, as well as reduce the total analysis time, despite the use of prolonged shallow gradient elution.
AB - We successfully identified the proteome expressed in Escherichia coli (E. coli) cells on a microarray scale using one-dimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a 350 cm long, 100 μm i. d., monolithic silica-C18 capillary column. E. coli tryptic digest (4 μg) was injected onto the column, and a 41 h gradient was applied with a flow rate of 500 nL/min at less than 20 MPa. In total, 22 196 nonredundant tryptic peptides from 2602 proteins, including 830 membrane proteins, were identified from the E. coli cells (triplicate analysis), in which an equivalent number of genes was detected by transcriptome analysis. Approximately a 5-fold larger peak response on average was obtained in this system, compared with that obtained by conventional capillary LC-MS/MS analysis with a 15 cm long, 3 μm diameter C18 silica particle-packed column. The higher response suggests that the influence of ionization suppression was drastically reduced by the high-efficiency separation on the long monolithic silica column coupled with the shallow gradient. Because this high-resolution system does not require any additional separation prior to LC-MS/MS, this "one-shot" proteomics approach can simplify the workflow of shotgun proteomics and minimize the sample amount, as well as reduce the total analysis time, despite the use of prolonged shallow gradient elution.
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U2 - 10.1021/ac100343q
DO - 10.1021/ac100343q
M3 - Article
C2 - 20222674
AN - SCOPUS:77950420818
VL - 82
SP - 2616
EP - 2620
JO - Industrial And Engineering Chemistry Analytical Edition
JF - Industrial And Engineering Chemistry Analytical Edition
SN - 0003-2700
IS - 7
ER -