Despite being orthogonal to reverse-phase separation and valuable for posttranslational modification (PTM) pre-enrichment, hydrophilic interaction liquid chromatography (HILIC) has not been widely adopted for large-scale proteomic applications. Here, we first evaluated the performance of HILIC in comparison with the popular high-pH reverse-phase (HPRP) separation, as the first dimension for tryptic peptide fractionation in a shotgun workflow to characterize the complex 293T cell proteome. The data indicated that the complementary nature of HILIC and HPRP for peptide separation was mainly due to different hydrophobicity preferences. Realizing that uncaptured components from one mode can be resolved in the other mode, we then designed and compared two multidimensional separation schemes using HILIC and HPRP in tandem for peptide prefractionation, in terms of identification efficiency and coverage at peptide, protein, and PTM levels. A total of 22,604 and 23,566 peptides corresponding to 4481 and 4436 proteins from 293T cell lysate were detected using HILIC-HPRP- and HPRP-HILIC-based shotgun proteomics workflow, respectively. In addition, without assistance of enrichment techniques, the tandem fractionation methods aided to identify 46 different PTMs from over 10,000 of spectra using blind modification search algorithm. We concluded that HILIC is a valuable alternative option for peptide prefractionation in a large-scale proteomic study, but can be further augmented with the use of a secondary HPRP separation.
Liquid chromatography coupled with tandem mass spectrometry
Multidimensional liquid chromatography
Reverse-phase liquid chromatography
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We thank Ms. Jing Jiang for LC-MS technical support.
This work was supported by The National Key Research and Development Program (2017YFC1200104), National Natural Science Foundation of China (81400589), Independent Project Fund of the State Key Laboratory for Diagnosis and Treatment of Infectious Disease, and the Zhejiang Provincial Medicine and Health Science and Technology Project (2016147735).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of MedicineZhejiang UniversityHangzhouChina