1. Liquid chromatography-tandem mass spectrometry approach for quantification of mucins from sputum using 13C,15N-labeled peptides as internal standards.
- Author
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Lindberg C, van Geest M, Lindberg H, and Kjellström S
- Subjects
- Amino Acid Sequence, Calibration, Carbon Isotopes chemistry, Humans, Isotope Labeling, Mucin 5AC standards, Mucin-5B standards, Nitrogen Isotopes chemistry, Chromatography, High Pressure Liquid standards, Mucin 5AC analysis, Mucin-5B analysis, Peptides chemistry, Sputum metabolism, Tandem Mass Spectrometry standards
- Abstract
Mucins are of great interest owing to their involvement in physiological and pathological processes in the airways. A method that allows accurate quantification of such proteins in sputum samples may be helpful for research in this field. A liquid chromatographic selected 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 C18 sorbent was used for sample cleanup prior to the liquid chromatography-tandem mass spectrometry (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 to 40 pmol/L. Both mucins could be determined with a precision of 6 to 19% and an accuracy of 98 to 114%. The method is transferable to robotics and is suitable to be run in a 96-well format., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2013
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