title: Application of microfluidic devices to proteomics research: identification of trace-level protein digests and affinity capture of target peptides.
abstract: This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 microl total volume), an array of separation channels, together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C(18) reverse phase packing or other type of affinity media such as immobilized antibodies or immobilized metal affinity chromatography beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration, and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per h and a concentration detection limit of approximately 5 nM (25 fmol on chip). Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2-1.8%, whereas relative standard deviation ranging from 9.2 to 11.8% are observed on peak heights. The application of this device for trace-level protein identification is demonstrated for two-dimensional gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint data base searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc-specific antibodies immobilized on protein G-Sepharose beads and facilitates the identification of antigenic peptides spiked at a level of 20 ng/ml in human plasma. Affinity selection is also demonstrated for gel-isolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system.
authors.affiliation: Institute for Biological Sciences, 100 Sussex Dr., Ottawa, Ontario K1A 0R6, Canada.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Li
authors.forename: Tammy
authors.initials: T
authors.lastname: LeRiche
authors.forename: Tammy-Lynn
authors.initials: TL
authors.lastname: Tremblay
authors.forename: Can
authors.initials: C
authors.lastname: Wang
authors.forename: Eric
authors.initials: E
authors.lastname: Bonneil
authors.forename: D Jed
authors.initials: DJ
authors.lastname: Harrison
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008297:Male; D008832:Microchemistry / Q000295:instrumentation*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D010449:Peptide Mapping; D011471:Prostatic Neoplasms / Q000737:chemistry / Q000235:genetics; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin; D014407:Tumor Cells, Cultured
chemical_list: D009363:Neoplasm Proteins; D010446:Peptide Fragments; D020543:Proteome; D016271:Proto-Oncogene Proteins c-myc; D014357:Trypsin