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Inventory of ‘slow exchanging’ hydrogen atoms in human proinsulin and its derivatives: observations on the mass spectrometric analysis of deuterio-proteins in D2O

Inventory of ‘slow exchanging’ hydrogen atoms in human proinsulin and its derivatives: observations on the mass spectrometric analysis of deuterio-proteins in D2O
Inventory of ‘slow exchanging’ hydrogen atoms in human proinsulin and its derivatives: observations on the mass spectrometric analysis of deuterio-proteins in D2O
Secondary structure elements of human proinsulin and of its tryptic products were compared by H/D exchange, in a single-pot, using mass spectrometry. Human proinsulin containing an N-terminal methionine, M-proinsulin, was engineered and converted into a perdeuterio derivative, which using an optimized mass spectrometric protocol and manual calculations gave a mass of 9669.6 (± 1) Da showing the replacement, with deuterium of 146.4 from a total of 149 exchangeable hydrogen atoms (83 from amides and 66 from side-chains). Tryptic digestion of the perdeuterio-M-proinsulin, followed by the transfer of the digest from a deuterio- into a protio-medium showed, at the earliest time of analysis, that of the 27 (± 1) D atoms retained in M-proinsulin, 24 (± 1) were found in the insulin nucleus, M-insulin-RR, and 4.2 (± 1) in the C-peptide-KR. A temporal analysis of the fate of D atoms in these species showed that whereas the C-peptide-KR rapidly exchanged its deuterium, losing all by 6 h, the loss of D atoms from M-proinsulin and M-insulin-RR was gradual and in each case, 12 deuterium atoms survived exchange for 72 h. At all time intervals the loss of D atoms from M-proinsulin mirrored that from M-insulin-RR plus the C-peptide-KR, suggesting that the secondary-structure elements of M-proinsulin are largely conserved in its two component parts.
proinsulin expression, proinsulin refolding, proinsulin HPLC, proinsulin mass spectrometry and x-ray diffraction, perdeuteriation of proinsulin, perdeuteriation of insulin, perdeuteriation of C-peptide and synthetic peptides, mass determination of perdeuterio-proteins, proinsulin-insulin structure comparison
1570-9639
1224-1233
Gardner, Qurra-tul-Ann Afza
6b69c9b8-b0c3-45ee-b7b8-43fcdaff9567
Younas, Hooria
cd0f1b06-520b-46a6-b729-d829d178d3f2
Rashid, Naeem
81704b5b-8999-4883-8e58-0c09ab9b63e1
Wright, J. Neville
e53ee4b9-10f5-4365-a9f2-5a7b0eacd86d
Akhtar, Muhammad
a4174002-a6bd-4678-8e63-4cbe607a672d
Gardner, Qurra-tul-Ann Afza
6b69c9b8-b0c3-45ee-b7b8-43fcdaff9567
Younas, Hooria
cd0f1b06-520b-46a6-b729-d829d178d3f2
Rashid, Naeem
81704b5b-8999-4883-8e58-0c09ab9b63e1
Wright, J. Neville
e53ee4b9-10f5-4365-a9f2-5a7b0eacd86d
Akhtar, Muhammad
a4174002-a6bd-4678-8e63-4cbe607a672d

Gardner, Qurra-tul-Ann Afza, Younas, Hooria, Rashid, Naeem, Wright, J. Neville and Akhtar, Muhammad (2009) Inventory of ‘slow exchanging’ hydrogen atoms in human proinsulin and its derivatives: observations on the mass spectrometric analysis of deuterio-proteins in D2O. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, 1794 (8), 1224-1233. (doi:10.1016/j.bbapap.2009.03.007). (PMID:19328246)

Record type: Article

Abstract

Secondary structure elements of human proinsulin and of its tryptic products were compared by H/D exchange, in a single-pot, using mass spectrometry. Human proinsulin containing an N-terminal methionine, M-proinsulin, was engineered and converted into a perdeuterio derivative, which using an optimized mass spectrometric protocol and manual calculations gave a mass of 9669.6 (± 1) Da showing the replacement, with deuterium of 146.4 from a total of 149 exchangeable hydrogen atoms (83 from amides and 66 from side-chains). Tryptic digestion of the perdeuterio-M-proinsulin, followed by the transfer of the digest from a deuterio- into a protio-medium showed, at the earliest time of analysis, that of the 27 (± 1) D atoms retained in M-proinsulin, 24 (± 1) were found in the insulin nucleus, M-insulin-RR, and 4.2 (± 1) in the C-peptide-KR. A temporal analysis of the fate of D atoms in these species showed that whereas the C-peptide-KR rapidly exchanged its deuterium, losing all by 6 h, the loss of D atoms from M-proinsulin and M-insulin-RR was gradual and in each case, 12 deuterium atoms survived exchange for 72 h. At all time intervals the loss of D atoms from M-proinsulin mirrored that from M-insulin-RR plus the C-peptide-KR, suggesting that the secondary-structure elements of M-proinsulin are largely conserved in its two component parts.

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Published date: August 2009
Keywords: proinsulin expression, proinsulin refolding, proinsulin HPLC, proinsulin mass spectrometry and x-ray diffraction, perdeuteriation of proinsulin, perdeuteriation of insulin, perdeuteriation of C-peptide and synthetic peptides, mass determination of perdeuterio-proteins, proinsulin-insulin structure comparison
Organisations: Centre for Biological Sciences

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Local EPrints ID: 335630
URI: http://eprints.soton.ac.uk/id/eprint/335630
ISSN: 1570-9639
PURE UUID: 6304e7ca-5c58-4def-b378-c6370aceaba6

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Date deposited: 13 Mar 2012 13:03
Last modified: 14 Mar 2024 10:37

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Contributors

Author: Qurra-tul-Ann Afza Gardner
Author: Hooria Younas
Author: Naeem Rashid
Author: J. Neville Wright
Author: Muhammad Akhtar

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