Combining solid phase extraction with ultra-short gradients in LC-MS/MS substantially improves protein identification per unit of time

ABSTRACT: Here, we describe a novel LC system enabling rapid protein/proteome analysis which requires <10 min analysis time. The setup consists of an autosampler harbouring two sets of 96 STAGE-tips which function as pre-columns, and a short RP analytical column, which can run a 5 min gradient. We show that the system allows the efficient combination with several popular off-line pre-fractionation methods, such as gel-electrophoresis, strong-cation exchange chromatography, but also in the analysis of interactomes obtained by affinity purification. A typical shotgun proteomics experiment, involving 36 SCX fractions of an AspN digested cell lysate, lead to the detection of over 3600 protein groups in only 3h cumulative gradient time when using a QExactive mass analyser. Application to typical various proteomics experiments such as samples from pull-downs allowed the detection of hundreds of proteins whereby a given protein’s interactome could be determined in less than 1h of accumulated gradient time. This new LC system diminishes proteome analysis time considerably, whole providing sufficient proteomic detail, and may thus also aid in bringing proteomics into the clinic where fast turn-over times are essential.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: N. Binai  

LAB HEAD: Albert Heck

PROVIDER: PXD001111 | Pride | 2014-12-12

REPOSITORIES: Pride