High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis
| Autor: | Kenneth J. Smith, Kellye A. Cupp-Sutton, Si Wu, Thomas Welborn, Mulin Fang, Zhe Wang |
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| Rok vydání: | 2020 |
| Předmět: |
Lysis
Protein digestion Hydrogen Deuterium Exchange-Mass Spectrometry 02 engineering and technology Mass spectrometry 01 natural sciences Biochemistry High-performance liquid chromatography Article Analytical Chemistry Escherichia coli Environmental Chemistry Spectroscopy Chromatography High Pressure Liquid Chromatography Chemistry Elution Protein dynamics 010401 analytical chemistry Temperature Deuterium Exchange Measurement 021001 nanoscience & nanotechnology 0104 chemical sciences Deuterium Hydrogen–deuterium exchange 0210 nano-technology Hydrogen |
| Zdroj: | Anal Chim Acta |
| ISSN: | 1873-4324 |
| Popis: | 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 °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 °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. |
| Databáze: | OpenAIRE |
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