期刊
ANALYTICA CHIMICA ACTA
卷 1143, 期 -, 页码 65-72出版社
ELSEVIER
DOI: 10.1016/j.aca.2020.11.022
关键词
HDX-MS; UPLC separation; Proteomics; High-throughput; Sub-zero temperature
资金
- NIH NIAID [R01 AI41625]
- NIH [NIH/NIAID-2U19AI062629]
HDX-MS is a powerful technique for studying protein dynamics and interactions, with recent technological advancements allowing for its application in analyzing complex protein samples. Optimized long gradient subzero-temperature UPLC separation conditions identified a large number of deuterated peptides in E. coli cell lysate digest.
Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein dynamics and protein interactions. Recent technological developments in the HDX-MS field, such as sub-zero LC separations, large-scale data analysis tools, and efficient protein digestion methods, have allowed for the application of HDX-MS to the analysis of multi protein systems in addition to pure protein analysis. Still, high-throughput HDX-MS analysis of complex samples is not widespread because the co-elution of peptides combined with increased peak complexity after labeling makes peak de-convolution extremely difficult. Here, for the first time, we evaluated and optimized long gradient subzero-temperature ultra-high-pressure liquid chromatography (UPLC) separation conditions for the HDX-MS analysis of complex protein samples such as E. coli. cell lysate digest. Under the optimized conditions, we identified 1419 deuterated peptides from 320 proteins at -10 degrees C, which is about 3-fold more when compared with a 15-min gradient separation under the same conditions. Interestingly, our results suggested that the peptides eluted late in the gradient are well-protected by peptide-column interactions at -10 degrees C so that peptides eluted even at the end of the gradient maintain high levels of deuteration. Overall, our study suggests that the optimized, sub-zero, long-gradient UPLC separation is capable of characterizing thousands of peptides in a single HDX-MS analysis with low back-exchange rates. As a result, this technique holds great potential for characterizing complex samples such as cell lysates using HDX-MS. Published by Elsevier B.V.
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