Ultratrace LC/MS proteomic analysis using 10-microm-i.d. Porous layer open tubular poly(styrene-divinylbenzene) capillary columns.
In this paper, the preparation and performance of long, high-efficiency poly(styrene-divinylbenzene) (PS-DVB), 10-microm-i.d. porous layer open tubular (PLOT) capillary columns are described. PLOT capillaries ( approximately 3% RSD column-to-column retention time), with relatively high permeability, were prepared by in-situ polymerization. Relatively high loading capacities, approximately 100 fmol for angiotensin I and approximately 50 fmol for insulin, were obtained with a 4.2 m x 10-microm-i.d. PLOT column. Low detection levels (attomole to sub-attomole) were achieved when the column was coupled on-line with a linear ion trap MS (LTQ). Analysis of human epidermal growth factor receptor (EGFR), a large transmembrane tyrosine kinase receptor with heterogeneous phosphorylation and glycosylation structures, was obtained at the 25 fmol level. The PLOT column yielded a peak capacity of approximately 400 for the separation of a complex tryptic digest mixture when the sample preparation included a 50-microm-i.d. PS-DVB monolithic precolumn and ESI-MS detection. As an example of the power of the column, 3046 unique peptides covering 566 distinct Methanosarcina acetivorans proteins were identified from a 50 ng in-gel tryptic digest sample combining five cuts in a single LC/MS/MS analysis using the LTQ. The results demonstrate the potential of the PLOT column for high-resolution LC/MS at the ultratrace level.
Yue
Guihua
G
Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
Luo
Quanzhou
Q
Zhang
Jian
J
Wu
Shiaw-Lin
SL
Karger
Barry L
BL
D019843:Archaeal Proteins / Q000032:analysis*; D002853:Chromatography, Liquid / Q000295:instrumentation*; D066246:ErbB Receptors / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D017020:Methanosarcina; D010766:Phosphorylation; D011137:Polystyrenes; D016062:Porosity; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
D019843:Archaeal Proteins; D011137:Polystyrenes; C003771:Amberlite XAD-2 resin; D066246:ErbB Receptors