Spectral counting assessment of protein dynamic range in cerebrospinal fluid following depletion with plasma-designed immunoaffinity columns

Abstract

BackgroundIn cerebrospinal fluid (CSF), which is a rich source of biomarkers for neurological diseases, identification of biomarkers requires methods that allow reproducible detection of low abundance proteins. It is therefore crucial to decrease dynamic range and improve assessment of protein abundance.ResultsWe applied LC-MS/MS to compare the performance of two CSF enrichment techniques that immunodeplete either albumin alone (IgYHSA) or 14 high-abundance proteins (IgY14). In order to estimate dynamic range of proteins identified, we measured protein abundance with APEX spectral counting method.Both immunodepletion methods improved the number of low-abundance proteins detected (3-fold for IgYHSA, 4-fold for IgY14). The 10 most abundant proteins following immunodepletion accounted for 41% (IgY14) and 46% (IgYHSA) of CSF protein content, whereas they accounted for 64% in non-depleted samples, thus demonstrating significant enrichment of low-abundance proteins. Defined proteomics experiment metrics showed overall good reproducibility of the two immunodepletion methods and MS analysis. Moreover, offline peptide fractionation in IgYHSA sample allowed a 4-fold increase of proteins identified (520 vs. 131 without fractionation), without hindering reproducibility.ConclusionsThe novelty of this study was to show the advantages and drawbacks of these methods side-to-side. Taking into account the improved detection and potential loss of non-target proteins following extensive immunodepletion, it is concluded that both depletion methods combined with spectral counting may be of interest before further fractionation, when searching for CSF biomarkers. According to the reliable identification and quantitation obtained with APEX algorithm, it may be considered as a cheap and quick alternative to study sample proteomic content.

Chemoproteomic Analysis of Intertissue and Interspecies Isoform Diversity of AMP-activated Protein Kinase (AMPK)

“…Likewise, ␤2 was the major isoform in human skeletal muscle but a minor isoform in human heart. Spectral counting has widely been utilized for the relative quantification of proteins in multiple biological samples (37)(38)(39)(40)(41)(42)(43)(44)(45)(46), but its application to the relative quantification of isoforms remains to be validated. In theory, the respective peak intensities of highly homologous peptides should be an ideal measure of the relative abundance of subunit isoforms provided that these peptides are co-eluted and identified in the same gel slice.…”

“…Relative abundance of the subunit isoforms was assessed by comparing the "spectral counts" of the isoform-unique peptides (Table 1) (37)(38)(39)(40)(41)(42)(43)(44)(45)(46). For instance, in the sample designated healthy hepatocyte-1, ␤1 and ␤2 isoforms were both identified with spectral counts of 6 and 92 (1:15), respectively.…”

Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid

“…Because of the greatly decreased protein concentration of this biofluid, the application of immunoaffinity depletion to this sample type has been largely limited to large samples (>1 mL) [32, 41, 46]. In studies with smaller mammals, such as mice where <100 μL of CSF can be obtained [47], immunoaffinity methods become unworkable.…”

Identifying and Overcoming Matrix Effects in Drug Discovery and Development

“…Recently, significant efforts have been devoted to the development of LC-MS/MS-based biomarker assays for diagnostic purposes (Ahmed et al, 2003;Apweiler et al, 2009;Pusch et al, 2003). Affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-page) can be utilized to deplete plasma samples of highly abundant proteins and improve detection limits of biomarkers (Ahmed et al, 2003;Borg et al, 2011;Liu et al, 2009). …”