Abstract
Despite many recent advances in instrumentation, the sheer complexity of biological samples remains a major challenge in large-scale proteomics experiments, reflecting both the large number of protein isoforms and the wide dynamic range of their expression levels. However, while the dynamic range of expression levels for different components of the proteome is estimated to be ∼107–8, the equivalent dynamic range of LC–MS is currently limited to ∼106. Sample pre-fractionation has therefore become routinely used in large-scale proteomics to reduce sample complexity during MS analysis and thus alleviate the problem of ion suppression and undersampling. There is currently a wide range of chromatographic techniques that can be applied as a first dimension separation. Here, we systematically evaluated the use of hydrophilic interaction liquid chromatography (HILIC), in comparison with hSAX, as a first dimension for peptide fractionation in a bottom-up proteomics workflow. The data indicate that in addition to its role as a useful pre-enrichment method for PTM analysis, HILIC can provide a robust, orthogonal and high-resolution method for increasing the depth of proteome coverage in large-scale proteomics experiments. The data also indicate that the choice of using either HILIC, hSAX, or other methods, is best made taking into account the specific types of biological analyses being performed.
Highlights
It is not so long since researchers would have counted themselves lucky to identify a few tens of proteins from a single shotgun proteomics experiment
Horie et al described the use of a meter-scale monolithic silica capillary column modified with urea functional groups for use in the hydrophilic interaction liquid chromatography (HILIC) mode, which provided highly orthogonal separation to RPLC with sufficient peak capacity, as well as highly sensitive detection for tryptic peptides
They reported on average ∼5-fold increase in the peak response for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase suggesting its application as a complementary tool to increase proteome coverage in proteomics studies [44]
Summary
It is not so long since researchers would have counted themselves lucky to identify a few tens of proteins from a single shotgun proteomics experiment. Yang et al [43] have evaluated the different stationary phases used in HILIC, addressing the effect of mobile phase composition on peak efficiencies with an online HILIC-ES-MS system using peptide mixtures and protein digests This showed that the use of HILIC-ES-MS provided complementary separation selectivity to RPLC-ES-MS and offered the capability to identify unique peptides, highlighting its potential in proteomic applications. Horie et al described the use of a meter-scale monolithic silica capillary column modified with urea functional groups for use in the HILIC mode, which provided highly orthogonal separation to RPLC with sufficient peak capacity, as well as highly sensitive detection for tryptic peptides In effect, they reported on average ∼5-fold increase in the peak response for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase suggesting its application as a complementary tool to increase proteome coverage in proteomics studies [44]. We systematically evaluate the performance of HILIC against the popular, hydrophilic strong anion exchange (hSAX) method of peptide fractionation, which separates peptides based on their charge [45]
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