Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation

Protein Science - Tập 2 Số 4 - Trang 522-531 - 1993
Zhongqi Zhang1, David L. Smith
1Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.

Tóm tắt

AbstractA new method based on protein fragmentation and directly coupled microbore high‐performance liquid chromatography–fast atom bombardment mass spectrometry (HPLC‐FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2–3, 0 °C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC‐FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h−1) to very slow (k < 0.002 h−1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC‐FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC‐FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.

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Tài liệu tham khảo

10.1016/S0022-2836(83)80235-6

10.1021/bi00385a032

10.1016/0022-2836(90)90200-6

10.1016/0003-2697(86)90035-7

10.1016/0022-2836(87)90613-9

10.1016/0003-2697(79)90693-6

10.1016/0003-2697(85)90033-8

10.1126/science.256.5064.1684

10.1017/S0033583500005217

10.1002/bip.1969.360070309

10.1016/S0065-3233(08)60129-1

10.1021/bi00498a001

10.1038/296713a0

10.1021/bi00354a036

10.1021/bi00752a003

10.1021/bi00243a025

10.1021/bi00842a035

10.1021/bi00542a030

10.1016/0076-6879(82)88084-1

10.1126/science.1697101

10.1038/335700a0

10.1021/bi00346a055

10.1021/bi00346a056

10.1016/0022-2836(79)90400-5

Smith D.L., 1990, Continuous‐Flow Fast Atom Bombardment Mass Spectrometry, 137

10.1002/pro.5560010506

10.1073/pnas.77.11.6371

10.1021/ac00044a027

10.1021/bi00399a008

10.1016/0022-2836(82)90180-2

10.1021/bi00353a025

10.1007/BF00421225

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Protein Science

Tập 2 Số 4

522-531

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