Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies

Abstract

Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to 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 Convective Interaction Media analytical columns (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 in vitro fertilization 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 nondepleted 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 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively.

Liquid‐phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in‐depth proteomics analysis—An update covering the period 2014–2016

“…A recent report described a novel immunoaffinity‐based convective interaction media analytical column (CIMac) with specificity to HSA (CIMac‐αHSA) for the fast and automated protein depletion . The polymethacrylate CIMac monolithic column allowed relatively shorter analysis time than the traditional chromatographic supports.…”

Use of monolithic supports for high‐throughput protein and peptide separation in proteomics

“…Depletion of high‐abundance proteins is one possible strategy that enables, or at least improves, analysis of certain low abundant proteins. Recent study demonstrated removal up to 94% of HSA from cell culture media, utilized for clinical embryo growth, with monolithic column containing immobilized anti‐HSA Ab . Upon depletion of HSA, the number of identified cellular proteins in the analysed medium by MS increased 50%.…”

Chromatographic Monoliths for High-Throughput Immunoaffinity Isolation of Transferrin from Human Plasma

“…The CIMac-@Tf columns were successfully prepared according to procedure developed for immobilization of polyclonal antibody specific for human serum albumin [27] using a mAb specific for transferrin (@Tf) and a hydrazide column, as described in the paragraph Preparation of columns with immobilized @Tf. After antibody immobilization, we confirmed that the column is specific for Tf.…”