Journal
JOURNAL OF PROTEOME RESEARCH
Volume 12, Issue 6, Pages 2449-2457Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr301011r
Keywords
ultralow hydrophobicity; chromatography; proteomics; strong anion exchange; hydrophilic strong anion exchange; HPLC; mouse macrophage; RAW264.7; orthogonality; mass spectrometry
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Funding
- Medical Research Council (MRC) UK
- Scottish Institute for Cell Signaling (SCILLS)
- MRC [MC_UU_12016/5, G1100713, MC_UP_A500_1020] Funding Source: UKRI
- Medical Research Council [G1100713, MC_UP_A500_1020, MC_UU_12016/5] Funding Source: researchfish
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Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong,cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.
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