On-Line multitasking analytical proteomics: how to separate, reduce, alkylate and digest whole proteins in an on-Line multidimensional chromatography system coupled to MS.
An on-Line multidimensional system has been developed, consisting of pH gradient strong anion exchange chromatography of native proteins in the first dimension with subsequent trapping and on-column reduction/alkylation on C4 trap columns and RP separation of the alkylated proteins in the second dimension followed by on-column tryptic digestion and electrospray MS detection. The system was evaluated using model proteins and a human urine sample. Compared to the commonly used in-solution alkylation method, the developed on-column method provides an equivalent efficiency. The recovery from the C4 trap columns of the alkylated proteins relative to the native state was from 94 to 102%. On-column tryptic digestion was satisfactory for many, but not for all proteins. The whole analytical procedure was performed on-Line with packed capillary columns for a total time of 320 min for the first ion exchange fraction, with additional 60 min for each subsequent fraction.
Tran
Bao Quoc
BQ
Department of Chemistry, University of Oslo, Blindern, Oslo, Norway. b.q.tran@kjemi.uio.no
Loftheim
Håvard
H
Reubsaet
Leon
L
Lundanes
Elsa
E
Greibrokk
Tyge
T
D000478:Alkylation; D000818:Animals; D002417:Cattle; D002645:Chickens; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D045304:Cytochromes c / Q000032:analysis; D004800:Enzymes, Immobilized / Q000737:chemistry; D006736:Horses; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000032:analysis; D009113:Muramidase / Q000032:analysis; D009211:Myoglobin / Q000032:analysis; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin / Q000737:chemistry; D014556:Urine / Q000737:chemistry
D004800:Enzymes, Immobilized; D007782:Lactoglobulins; D009211:Myoglobin; D011506:Proteins; D012709:Serum Albumin; D045304:Cytochromes c; D009113:Muramidase; D014357:Trypsin