A novel chromatographic method allows on-line reanalysis of the proteome.
Liquid chromatography combined with electrospray ionization is widely used for direct analysis of polar and labile molecules by LCMS. The on-line coupling in LCMS is a major strength but also causes a principal limitation that each eluting analyte has to be analyzed immediately and is not available for detailed interrogation after the LCMS run. Here we developed a new chromatographic strategy, which removes this limitation. After column separation the flow is split, one portion is analyzed directly, and the other is diverted to a capture capillary. After the direct LCMS run, the flow is switched, and the portion stored in the capillary is analyzed ("replay run"). We describe a setup consisting of an analytical column, a splitting valve, and a focusing column, which performs at full sensitivity and undiminished chromatographic resolution. We demonstrate three principal advantages of this system: nearly continuous MS utilization, duplicate analysis without requirement for additional sample, and targeting of important but undersampled features in the replay run.
Waanders
Leonie F
LF
Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Almeida
Reinaldo
R
Prosser
Simon
S
Cox
Jürgen
J
Eikel
Daniel
D
Allen
Mark H
MH
Schultz
Gary A
GA
Mann
Matthias
M
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D008099:Liver / Q000737:chemistry*; D051379:Mice; D020543:Proteome / Q000032:analysis*; D012680:Sensitivity and Specificity; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D020543:Proteome; C515972:SUMO2 protein, mouse; D025841:Small Ubiquitin-Related Modifier Proteins