Dataset Information

Displacement Chromatography Mode – High protein sequence coverages and identification rates for low µg-range proteomics using online 2D-LC-MS

ABSTRACT: In bottom-up proteomics, the complexity of proteomes is still a challenge. For deep proteome profiling, multi-dimensional separation strategies and large initial protein amounts are required. Here, we report on an online 2D-LC-MS/MS approach applying strong cation exchange chromatography (SCX) in displacement chromatography mode (DCM) as the first dimension of separation. This method enables a comprehensive analysis of proteomes in the low µg-range (5 µg). The first detailed comparison of DCM with conventionally used gradient chromatography mode (GCM) highlights a significantly better separation efficiency with DCM. Especially for peptides with a net-charge state (NCS) of +2, which represents the majority of tryptic peptides in mammalian proteomes, DCM provides a significantly better separation. These peptides were separated over several fractions in DCM, which is not possible to achieve in GCM due to theirs low affinity towards the SCX column. The better separation in DCM results not only in a considerably higher reproducibility (Pearson’s r=0.93), but significantly increases identification rates of both peptides and proteins. For peptides with a low NCS, DCM achieved a 2.6-fold increase in identified peptides. In total, DCM provides a 1.5-fold increase in peptide identifications and a 1.7-fold increase of protein identifications. The number of identified unique peptides per protein and the protein sequence coverages were significantly higher in DCM compared to GCM providing more reliable quantitative results. The higher sequence coverage are of interest for applications such as proteogenomics, where it is important to have high protein sequence coverages to identify single amino acid variants and splice-junction peptides.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Marcel Kwiatkowski