Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-microm-i.d. polystyrene-divinylbenzene monolithic capillary columns.
This work explores the use of 20-microm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100000 plates/m for peptide separations. High mass sensitivity (approximately 10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-microm-i.d. nanocolumns. A wide linear dynamic range (approximately 4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of approximately 1000 cells injected, is presented.
Ivanov
Alexander R
AR
Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, USA.
Zang
Li
L
Karger
Barry L
BL
D000595:Amino Acid Sequence; D000818:Animals; D018270:Carcinoma, Ductal, Breast / Q000737:chemistry; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002374:Catalase / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D030562:Databases, Protein; D005260:Female; D006736:Horses; D006801:Humans; D007782:Lactoglobulins / Q000737:chemistry / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D009479:Neuropeptides / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis*; D011137:Polystyrenes / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D014020:Tissue Extracts / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D014362:Trypsinogen / Q000737:chemistry / Q000378:metabolism
D002364:Caseins; D007782:Lactoglobulins; D009211:Myoglobin; D009479:Neuropeptides; D010446:Peptide Fragments; D011137:Polystyrenes; D011506:Proteins; D014020:Tissue Extracts; C432006:divinylbenzene-polystyrene copolymer; D014362:Trypsinogen; D010047:Ovalbumin; D045304:Cytochromes c; D002374:Catalase; D014357:Trypsin