Quantitative erythrocyte membrane proteome analysis with Blue-native/SDS PAGE.
The erythrocyte membrane plays a pivotal role in erythrocyte functioning. Many membrane protein aberrations are known that result in hemolytic anemia, however, the origin of numerous disorders is not known to date. To extend the current set of diagnostic tools, we used a novel proteome-wide approach to quantitatively analyze membrane proteins of healthy donor and patient erythrocytes. Blue-native PAGE has proven to be a powerful tool for separation of membrane proteins and their complexes, but has hitherto not been applied to erythrocyte membranes to find biomarkers. Using this technique, we detected almost 150 protein spots, from which more than 500 proteins could be identified by LC-MS/MS. Further, we successfully assessed the potential of using CyDye labeling to quantify the membrane proteins. Our final goal was to determine if this approach is suited to detect protein level changes in disordered erythrocyte membranes, and we could successfully confirm that erythrocyte spectrin levels were dramatically decreased for a hemolytic anemia patient. This approach provides a new tool to detect potential biomarkers and can contribute to an improved understanding of the causes of erythrocyte membrane defects in patients suffering from hemolytic anemia.
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, the Netherlands.
Renske A
RA
van Gestel
Wouter W
WW
van Solinge
H W P
HW
van der Toorn
Gert
G
Rijksen
Albert J R
AJ
Heck
Richard
R
van Wijk
Monique
M
Slijper
D000328:Adult; D000740:Anemia / Q000097:blood; D001782:Blood Donors; D002138:Calibration; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods / Q000592:standards; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods* / Q000592:standards; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes / Q000494:pharmacology; D006262:Health; D006801:Humans; D046912:Multiprotein Complexes / Q000032:analysis / Q000378:metabolism; D011489:Protein Denaturation / Q000502:physiology; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012394:Rosaniline Dyes / Q000494:pharmacology
D005456:Fluorescent Dyes; D046912:Multiprotein Complexes; D020543:Proteome; D012394:Rosaniline Dyes; C004692:coomassie Brilliant Blue