Tarasova, I. A., Lobas, A. A., Černigoj, U., Solovyeva, E. M., Mahlberg, B., Ivanov, M. V., Panić-Janković, T., Nagy, Z., Pridatchenko, M. L., Pungor, A., Nemec, B., Vidič, U., Gašperšič, J., Krajnc, N. L., Vidič, J., Gorshkov, M. V. and Mitulović, G. ELECTROPHORESIS. Accepted Author Manuscript. doi:10.1002/elps.201500489.
Affinity depletion of abundant proteins such as human serum albumin (HSA) is an important stage in routine sample preparation prior to tandem mass spectrometry (MS/MS) analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study we performed evaluation of a novel immunoaffinity-based CIMac depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate-independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and Cleavage/Blastocyst IVF culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100% ± 25% in albumin-enriched fractions relative to the non-depleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5 to 30% and 20 to 30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively.
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