Mass Spectrometry Method to Measure Membrane Proteins in Dried Blood Spots for the Detection of Blood Doping Practices in Sport.

ABSTRACT

The dried blood spot (DBS) matrix has significant utility for applications in the field where venous blood collection and timely shipment of labile blood samples is difficult. Unfortunately, protein measurement in DBS is hindered by high abundance proteins and matrix interference that increases with hematocrit. We developed a DBS method to enrich for membrane proteins and remove soluble proteins and matrix interference. Following a wash in a series of buffers, the membrane proteins are digested with trypsin and quantitated by parallel reaction monitoring mass spectrometry methods. The DBS method was applied to the quantification of four cell-specific cluster of differentiation (CD) proteins used to count cells by flow cytometry, band 3 (CD233), CD71, CD45, and CD41. We demonstrate that the DBS method counts low abundance cell types such as immature reticulocytes as well as high abundance cell types such as red blood cells, white blood cells, and platelets. When tested in 82 individuals, counts obtained by the DBS method demonstrated good agreement with flow cytometry and automated hematology analyzers. Importantly, the method allows longitudinal monitoring of CD protein concentration and calculation of interindividual variation which is difficult by other methods. Interindividual variation of band 3 and CD45 was low, 6 and 8%, respectively, while variation of CD41 and CD71 was higher, 18 and 78%, respectively. Longitudinal measurement of CD71 concentration in DBS over an 8-week period demonstrated intraindividual variation 17.1-38.7%. Thus, the method may allow stable longitudinal measurement of blood parameters currently monitored to detect blood doping practices.