title: Two-dimensional strong cation exchange/porous layer open tubular/mass spectrometry for ultratrace proteomic analysis using a 10 microm id poly(styrene- divinylbenzene) porous layer open tubular column with an on-line triphasic trapping column.
abstract: This study expands the capabilities for ultratrace proteomic analysis of our previous work by incorporating on-line sample desalting using a triphasic (RP/strong cation exchange (SCX)/micro-SPE) trapping column connected to a 3.2 m x 10 microm id poly(styrene-divinylbenzene) (PS-DVB) porous layer open tubular (PLOT) column. To minimize extra sample handling steps, C18 RP packing was incorporated in the capillary tubing upstream of the SCX column for the on-line desalting. For the micro-SPE column, a 50 microm id PS-DVB monolithic column was positioned downstream of the SCX column. High-performance separation was achieved on the PLOT column at a mobile phase flow rate of 20 nL/min. The sensitivity and high resolution capability of the new multidimensional platform was evaluated using an in-gel tryptic digested sample of a cervical cancer (SiHa) cell line. For the injected amount of 1200 cells ( approximately 500 ng), over 2700 peptides covering greater than 850 unique proteins were identified from the triphasic SCX/PLOT/MS analysis of a single SDS gel section (>40 kDa). The 2-D LC/MS platform demonstrated good separation performance, such that more than 85% of the identified peptides were detected from only one salt fraction. In a triplicate analysis of the above >40 kDa gel section, 4497 peptides and 1209 unique proteins were identified when applying stringent filtering criteria, with a false-positive rate of 2.4%. When all three SDS-PAGE gel sections of the lysed SiHa cells were analyzed, 5047 peptides and 1857 unique proteins (false-positive rate 1.8%), including cancer-related proteins such as MAP kinases, were identified.
authors.affiliation: Department of Chemistry and Chemical Biology, Barnett Institute, Northeastern University, Boston, MA 02115, USA.
authors.forename: Quanzhou
authors.initials: Q
authors.lastname: Luo
authors.forename: Ye
authors.initials: Y
authors.lastname: Gu
authors.forename: Shiaw-Lin
authors.initials: SL
authors.lastname: Wu
authors.forename: Tomas
authors.initials: T
authors.lastname: Rejtar
authors.forename: Barry L
authors.initials: BL
authors.lastname: Karger
mesh_terms: D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry / Q000379:methods*; D040901:Proteomics; D012680:Sensitivity and Specificity
chemical_list: D002411:Cation Exchange Resins