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Liquid chromatography-tandem mass spectrometry approach for quantification of mucins from sputum using C-13,N-15-labeled peptides as internal standards

Lindberg, Claes; van Geest, Marleen; Lindberg, Henrik and Kjellström, Sven LU (2013) In Analytical Biochemistry 434(1). p.84-92
Abstract
Mucins are of great interest owing their involvement in physiological and pathological processes in the airways. A method which allows accurate quantification of such proteins in sputum samples may be helpful for research in this field. A liquid chromatographic single reaction monitoring (SRM) method was developed for the quantification of two mucins, MUC5AC and MUC5B, in induced sputum samples. Sample preparation for the assay included solubilization, reduction and alkylation prior to tryptic digestion. Solid phase extraction using C(18) sorbent was used for sample clean up prior to the LC-MS/MS analysis. A cysteine containing peptide was selected for quantification of MUC5AC protein, whereas a non-cysteine peptide was used for the... (More)
Mucins are of great interest owing their involvement in physiological and pathological processes in the airways. A method which allows accurate quantification of such proteins in sputum samples may be helpful for research in this field. A liquid chromatographic single reaction monitoring (SRM) method was developed for the quantification of two mucins, MUC5AC and MUC5B, in induced sputum samples. Sample preparation for the assay included solubilization, reduction and alkylation prior to tryptic digestion. Solid phase extraction using C(18) sorbent was used for sample clean up prior to the LC-MS/MS analysis. A cysteine containing peptide was selected for quantification of MUC5AC protein, whereas a non-cysteine peptide was used for the quantification of MUC5B protein. Stable isotope-labeled synthetic peptides were used as internal standards and linear calibration curves were constructed in the range of 0.3 - 40 pmol/L. Both mucins could be determined with a precision of 6-19% and an accuracy of 98-114%. The method is transferable to robotics and is suitable to be run in a 96-well format. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Sputum, Mass spectrometry, Mucins
in
Analytical Biochemistry
volume
434
issue
1
pages
84 - 92
publisher
Elsevier
external identifiers
  • wos:000314378200015
  • pmid:23138178
  • scopus:84871693834
ISSN
1096-0309
DOI
10.1016/j.ab.2012.10.033
language
English
LU publication?
yes
id
ba989c42-eb70-415a-ac8c-35ae15152523 (old id 3219110)
date added to LUP
2012-12-14 12:21:49
date last changed
2019-01-06 04:17:55
@article{ba989c42-eb70-415a-ac8c-35ae15152523,
  abstract     = {Mucins are of great interest owing their involvement in physiological and pathological processes in the airways. A method which allows accurate quantification of such proteins in sputum samples may be helpful for research in this field. A liquid chromatographic single reaction monitoring (SRM) method was developed for the quantification of two mucins, MUC5AC and MUC5B, in induced sputum samples. Sample preparation for the assay included solubilization, reduction and alkylation prior to tryptic digestion. Solid phase extraction using C(18) sorbent was used for sample clean up prior to the LC-MS/MS analysis. A cysteine containing peptide was selected for quantification of MUC5AC protein, whereas a non-cysteine peptide was used for the quantification of MUC5B protein. Stable isotope-labeled synthetic peptides were used as internal standards and linear calibration curves were constructed in the range of 0.3 - 40 pmol/L. Both mucins could be determined with a precision of 6-19% and an accuracy of 98-114%. The method is transferable to robotics and is suitable to be run in a 96-well format.},
  author       = {Lindberg, Claes and van Geest, Marleen and Lindberg, Henrik and Kjellström, Sven},
  issn         = {1096-0309},
  keyword      = {Sputum,Mass spectrometry,Mucins},
  language     = {eng},
  number       = {1},
  pages        = {84--92},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Liquid chromatography-tandem mass spectrometry approach for quantification of mucins from sputum using C-13,N-15-labeled peptides as internal standards},
  url          = {http://dx.doi.org/10.1016/j.ab.2012.10.033},
  volume       = {434},
  year         = {2013},
}