Sample complexity reduction aids efficient detection of low-abundant proteins from human amniotic fluid.
Working with biological fluids poses a challenge of visualizing proteins present in lower concentrations. This study describes a batch-mode chromatographic method for the fractionation of human amniotic fluid (AF). This method is easy to use with minimal sample quantity, resin volume and sample processing time. For albumin depletion, two methods were evaluated. The results demonstrated that specific depletion of albumin, using affinity-ligand-based resin, is more efficient than the conventional dye-based method. The albumin-depleted human AF was fractionated by strong anion-exchange resin in spin devices, for sample, complexity reduction and enrichment of low-abundant proteins. Analysis of four eluate fractions generated after this step shows enrichment of few low-abundant proteins. Two novel low-abundant proteins, Rab GDP dissociation inhibitor beta and peptide methionine sulfoxide reductase, were identified from human AF. Alpha-1-B glycoprotein was successfully identified by this strategy, whereas the published literature reports that it was not identified by strong anion-exchange FPLC followed by SDS-PAGE. Therefore, the current method has distinct advantages over the conventional column-based chromatography. This study also reports altered expression of some proteins in Rh-isoimmunized AF samples in comparison with normal AF.
Alam
Mashkoor
M
Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India.
Selladurai
Masilamani
M
Nagpal
Saurabh
S
Tomar
Anil Kumar
AK
Saraswat
Mayank
M
Raziuddin
M
M
Mittal
Sunita
S
Singh
Tej P
TP
Yadav
Savita
S
D000653:Amniotic Fluid / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D011506:Proteins / Q000032:analysis*
D011506:Proteins