Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 877; no. 8; pp. 663 - 670
• Main Authors: Zhao, Rui, Ding, Shi-Jian, Shen, Yufeng, Camp II, David G., Livesay, Eric A., Udseth, Harold, Smith, Richard D.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.03.2009; Elsevier
• Subjects: Amino Acid Sequence; Analysis; Analytical, structural and metabolic biochemistry; Animals; Automation; Biological and medical sciences; Cells, Cultured; Cercopithecus aethiops; COS Cells; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Mass spectrometer; Mass Spectrometry - instrumentation; Mass Spectrometry - methods; Medical sciences; Metal free nano-LC; Molecular Sequence Data; On line SPE column; Pharmacology. Drug treatments; Phosphopeptide; Phosphopeptides - chemistry; Sensitivity and Specificity; On line SPE column; Automation; Metal free nano-LC; Mass spectrometer; Phosphopeptide; Solid phase extraction; Peptides; Metal; Chemical enrichment; Sensitivity; Sample preparation; Analysis; Miniaturization; Mass spectrometry
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2008.12.068

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 μm i.d. × 30 cm containing 5 μm C18 particles) and the on-line solid phase extraction columns (150 μm i.d. × 4 cm containing 5 μm 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 ∼250 for phosphopeptides (and ∼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-μm i.d. column of the nanoLC. The separation power and sensitivity provided by the nanoLC–LTQ enabled identification of ∼4600 phosphopeptide candidates from ∼60 μg COS-7 cell tryptic digest followed by IMAC enrichment and ∼520 tyrosine phosphopeptides from ∼2 mg of human T cells digests followed by phosphotyrosine peptide immunoprecipitation.