Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput.
We report on the development and characterization of automated metal-free multiple-column nanoLC instrumentation for sensitive and high-throughput analysis of phosphopeptides with mass spectrometry. The system employs a multiple-column capillary LC fluidic design developed for high-throughput analysis of peptides (Anal. Chem. 2001, 73, 3011-3021), incorporating modifications to achieve broad and sensitive analysis of phosphopeptides. The integrated nanoLC columns (50 microm i.d. x 30 cm containing 5 microm C18 particles) and the on-line solid phase extraction columns (150 microm i.d. x 4 cm containing 5 microm C18 particles) were connected to automatic switching valves with non-metal chromatographic accessories, and other modifications to avoid the exposure of the analyte to any metal surfaces during handling, separation, and electrospray ionization. The nanoLC developed provided a separation peak capacity of approximately 250 for phosphopeptides (and approximately 400 for normal peptides). A detection limit of 0.4 fmol was obtained when a linear ion trap tandem mass spectrometer (Finnegan LTQ) was coupled to a 50-microm i.d. column of the nanoLC. The separation power and sensitivity provided by the nanoLC-LTQ enabled identification of approximately 4600 phosphopeptide candidates from approximately 60 microg COS-7 cell tryptic digest followed by IMAC enrichment and approximately 520 tyrosine phosphopeptides from approximately 2mg of human T cells digests followed by phosphotyrosine peptide immunoprecipitation.
Zhao
Rui
R
Environmental Molecular Sciences Laboratory and Biological Sciences Division, Pacific Northwest National Laboratory, P. O. Box 999, Richland, WA 99352, USA.
Ding
Shi-Jian
SJ
Shen
Yufeng
Y
Camp
David G
DG
Livesay
Eric A
EA
Udseth
Harold
H
Smith
Richard D
RD
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry*; D012680:Sensitivity and Specificity
D010748:Phosphopeptides