Integrated system for rapid proteomics analyses using microfluidic devices coupled to nanoelectrospray mass spectrometry.
This chapter presents an integrated and modular microsystem providing rapid analyses of low femtomole of in-gel digests for proteomics applications. Enhancement of sample throughput is facilitated using an autosampler, a microfabricated device comprising a large (2.4-microL total volume) separation channel together with a low-dead-volume interface to nES mass spectrometry. Sample preconcentration is achieved by packing C18 reverse phase or immobilized metal affinity chromatography (IMAC) beads into the large channel of this microfluidic device to adsorb peptides or enrich the sample in phosphopeptides prior to capillary electrophoresis separation and MS detection. This integrated microfluidic systems enables a sample throughput of up to 12 samples/h with a detection limit of approx 5 nM (25 fmol inj.). Replicate injections of peptide standards indicated that reproducibility of migration time was typically 1.2-1.8%, whereas relative standard deviation (RSD) values of 9.2-11.8% were obtained on peak heights. The application of this device is demonstrated for 2D gel spots obtained from protein extracts of human astrocyte cells and for excised bands of membrane proteins from Neisseria meningitidis. A stepped acetonitrile gradient can be incorporated with the present microfluidic system to enhance selectivity during sample analysis.
Li
Jianjun
J
Institute for Biological Sciences, Ottawa, Ontario, Canada.
Tremblay
Tammy-Lynn
TL
Harrison
Jed
J
Thibault
Pierre
P
D001253:Astrocytes / Q000737:chemistry*; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry*; D036103:Nanotechnology; D009345:Neisseria meningitidis / Q000737:chemistry; D040901:Proteomics; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D001426:Bacterial Proteins; D008565:Membrane Proteins