Application of online two-dimensional separation system using monolithic columns for proteome analysis of human cartilage

Abstract

In Shotgun proteome analysis, where nano-flow is adopted to increase the sensitivity as well as extremely complicated samples such as proteolytic digest are inevitably confronted, monolithic capillary columns are widely used to improve the liquid chromatography separation performance. It is known that cartilage contains extensive amounts of extracellular matrix (ECM), in which collagens and aggrecans being the most abundant macromolecules. It is obvious that the high content of ECM components causes a challenge in the comprehensive proteome analysis of cartilage. In this study, a 7 cm x 150 microm i. d. phosphate strong cation exchange (SCX) monolithic capillary column was coupled with an 85 cm x 75 microm i. d. C12 reversed-phase monolithic capillary column for online two-dimensional separation of 20 microg tryptic digest of proteins extracted from human cartilage. After 14 salt steps fractionation and following gradient separation coupled with tandem mass spectrometry detection, finally 7 434 unique peptides, corresponding to 1 901 distinct proteins were positively identified. Then, the identified proteins were analyzed by Gene Ontology (GO), and it was found that most of the identified proteins were come from articular chondrocytes with low abundance, which is important for the researches of articular diseases.