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

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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.

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Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Biologiska vetenskaper

Nyckelord

Originalspråkengelska
Sidor (från-till)84-92
TidskriftAnalytical Biochemistry
Volym434
Utgåva nummer1
StatusPublished - 2013
PublikationskategoriForskning
Peer review utfördJa