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Analysis of differential strategies to enhance detection of low abundance proteins in the bovine serum proteome

ABSTRACT: Serum-based biomarkers hold propitious applications for addressing livestock health, management, and welfare challenges. However, discovery of protein biomarkers in complex biological fluids like serum is wholly intractable due to the large dynamic range of protein concentrations; that is, ~10 abundant proteins constitute >90% of the total protein content. Consequently, the detection of lower-abundant serum proteins that may be valuable biomarkers is effectively masked in proteomic detection. To overcome this barrier, specific tools/methods have been developed for enriching and identifying low-abundance proteins/biomarkers in human samples. In species like cattle for which such tools do not exist, the detectable serum proteome remains seven-fold lower and biased towards high-abundance proteins, precluding possibilities of biomarker identification. Towards addressing this limitation, we test a continuous elution size-based fractionation method, and two approaches that use affinity interaction-based separation of proteins in preparing bovine serum, and compare LC-MS/MS protein identification to neat serum. Our results identify the high-abundance proteins in bovine serum, and demonstrate dynamic range compression and improved protein identification with the different enrichment methods. Although these findings indicate the highest protein number identified in bovine serum (444 proteins, all methods combined), and by any single sample processing method (312 proteins) till date, they remain lower than thresholds deemed necessary for biomarker discovery. As such, this investigation also revealed the limitations to resolving the bovine serum proteome, and the need for species-specific tools for depleting high-abundance proteins. In concert, this study represents a step towards advancing sample preparation methods for bovine serum biomarker identification.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Bos Taurus (ncbitaxon:9913)

SUBMITTER: Vimal Selvaraj

PROVIDER: MSV000085465 | MassIVE | Tue May 26 07:49:00 BST 2020

REPOSITORIES: MassIVE

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Project description:Detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures. We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168. Four of proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity. The method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify bacterial virulence factors, vaccine candidates and potential biomarkers. The overall study was done with five technical replicates. Ten proteins derived from Campylobacter jejuni were tested regarding their immunogenic potential. The ten proteins derived from Campylobacter jejuni were fusion proteins, i.e., N-terminal HaloTag fused to: Gene <--> Protein cjaA <--> putative amino-acid transporter periplasmic solute-binding protein hisJ <--> histidine-binding protein precursor pal <--> peptidoglycan associated lipoprotein flaC <--> flagellin flaA <--> flagellin peb1a <--> bifunctional adhesin/ABC transporter aspartate/glutamate-binding protein pyrC <--> dihydroorotase (EC:3.5.2.3) pseB <--> UDP-GlcNAc-specific C4,6 dehydratase/C5 epimerase gapA <--> glyceraldehyde 3-phosphate dehydrogenase (EC:1.2.1.12) argC <--> N-acetyl-gamma-glutamyl-phosphate reductase (EC:1.2.1.38)

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Analysis of differential strategies to enhance detection of low abundance proteins in the bovine serum proteome

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