Novel application of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) in shotgun proteomics: comprehensive profiling of rat kidney proteome.
In shotgun proteomics, multidimensional liquid chromatography (MDLC) is commonly used to reduce sample complexity and increase dynamic range of protein identification. Since reversed-phase chromatography is mostly used as the second-dimensional separation before mass spectrometric analysis, the improvement of MDLC primarily depends on the first dimension of separation. Here, we present a novel whole proteome analysis method that separates peptides based on ERLIC. Tryptic peptides were retained on a weak anion exchange column through ERLIC with a high organic mobile phase. They were then distributed into multiple fractions based on both pI and polarity through the simultaneous effect of electrostatic repulsion and hydrophilic interaction when eluted using a salt-free pH gradient of increasing water content. Applying this to rat kidney tissue, we identified 4821 proteins and 30 659 unique peptides with high confidence from two replicates using LTQ-FT. This was 36.2% and 64.3% higher, respectively, than was obtained with the widely used SCX separation mode. Notably, the identification of both highly hydrophobic and basic peptides increased over 120% using the ERLIC method. The results indicate that ERLIC is a promising alternative to SCX as the first dimension of MDLC. In total, 5499 proteins and 35 847 unique peptides of rat kidney tissue are characterized.
Hao
Piliang
P
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore.
Guo
Tiannan
T
Li
Xin
X
Adav
Sunil S
SS
Yang
Jie
J
Wei
Meng
M
Sze
Siu Kwan
SK
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D007526:Isoelectric Point; D007668:Kidney / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin