Reference Methods and Materials in Standardisation and Quality Assurance

Measurements in the health-related fields, like biomolecular protein marker determination are often seriously hampered by the lack of comparability of laboratory data. Contradictory measurement results are obtained due to a lack of traceability, commutability of reference materials (RMs), assay calibration and comparability of data. The role of reference measurement systems and reference materials in standardisation and quality assurance is addressed in this presentation. An example is given for the determination of myoglobin in human serum, introducing a new combined experimental and bioinformatics approach to reliably quantify the myoglobin content of serum samples. Quantification is achieved spiked amounts of myoglobin to a human serum sample. In addition, myoglobin was used as internal standard to account for fluctuations in peak areas due to sample preparation and mass spectrometric detection. Myoglobin was separated from the highly abundant serum proteins by means of strong anion-exchange chromatography. Subsequently, the myoglobin fraction was trypsinized and analyzed by high-performance liquid chromatography-electrospray ionization mass spectrometry. The raw data acquired by the instrument was analysed automatically using a newly developed algorithm that detects and quantifies all ions belonging to peptides in the sample. The developed algorithm then computes a linear regression and confidence interval for the series of additive measurements with and without internal standard. We could quantify the amount of myoglobin using several tryptic peptides in parallel in a completely automated fashion. The relative errors observed were as low as 2.5 %. This quantitative method could facilitate further absolute or relative quantitation of even more complex peptide samples, such as in the field of protein diagnostic markers.

KOHLBACHER Oliver;  HUBER Christian;  REINERT Knut; 

KLEIN Christoph; 

2005-12-07

ELSEVIER SCIENCE BV

JRC31292

https://publications.jrc.ec.europa.eu/repository/handle/JRC31292,