title: Liquid chromatography-tandem mass spectrometry approach for quantification of mucins from sputum using 13C,15N-labeled peptides as internal 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.
authors.affiliation: Biological Sciences, AstraZeneca R&D, 221 87 Lund, Sweden.
authors.forename: Claes
authors.initials: C
authors.lastname: Lindberg
authors.forename: Marleen
authors.initials: M
authors.lastname: van Geest
authors.forename: Henrik
authors.initials: H
authors.lastname: Lindberg
authors.forename: Sven
authors.initials: S
authors.lastname: Kjellström
mesh_terms: D000595:Amino Acid Sequence; D002138:Calibration; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D006801:Humans; D007553:Isotope Labeling; D055271:Mucin 5AC / Q000032:analysis* / Q000592:standards; D055257:Mucin-5B / Q000032:analysis* / Q000592:standards; D009587:Nitrogen Isotopes / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D013183:Sputum / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D002247:Carbon Isotopes; C524678:MUC5AC protein, human; C524578:MUC5B protein, human; D055271:Mucin 5AC; D055257:Mucin-5B; D009587:Nitrogen Isotopes; D010455:Peptides