Combinatorial peptide ligand library plasma treatment: Advantages for accessing low‐abundance proteins

Abstract

Depletion of major blood proteins is one of the most promising approaches to accessing low-abundance biomarkers. This study compared the use of combinatorial peptide ligand library (CPLL) and albumin and immunoglobulins (IgGs) depletion technology for accessing these low-abundance proteins in plasma using 2-DE in an acidic restricted pH range (4-7). Compared with native plasma, both techniques enlarge the visibility of other proteins than albumin and IgG, but there were marked differences in their composition. An increase of the number of spots was detected compared with native plasma (157 spots) with 427 and 557 spots, respectively, detected with albumin and IgG depletion, and CPLL treatment. We selected 70 spots to be identified by MALDI-TOF related to their absence in the 2-D gels from native or albumin and IgG-depleted plasma. The 42 spots identified corresponded to 24 different proteins, with more than half of the proteins which did not belong to the major plasma proteins. CPLL treatment allowed the accessibility to proteolytic fragments obtained from major plasma proteins. We found a large superiority of the CPLL approach over the albumin and IgG depletion process. These findings show the utility of depleting major blood proteins to be able to access low-abundance proteins and the potential of CPLL to select and identify candidate biomarkers in clinical studies.

Plasma-based proteomics reveals immune response, complement and coagulation cascades pathway shifts in heat-stressed lactating dairy cows

“…range of proteins present in plasma makes their analysis challenging, because high-abundance proteins tend to mask or interfere with the detection of proteins components present in small amounts [12]. Thus, depletion of these high-abundance plasma proteins prior to further analysis is a logical preprocessing step.…”

“…ProteoMiner enrichment kit (Bio-Rad, USA) utilizes a combinatorial library of hexapeptides rather than immunodepletion, minimizing the dependence on available antibodies and preventing the codepletion of low-abundance proteins [12]. In this study, we processed plasma samples with ProteoMiner enrichment kit (Bio-Rad, USA)…”

Low abundance protein enrichment for discovery of candidate plasma protein biomarkers for early detection of breast cancer

“…Other investigators have reported similar results. Beseme et al [32] and Sihlbom et al [18] compared the use of CPLL with albumin and IgG immunodepletion. These investigators detected an additional 30%-60% of protein spots on 2-D gels with CPLL.…”

“Proteomineering” serum biomarkers. A Study in Scarlet

“…The results are as follows: eluant 4 M urea11% CHAPS seems to elute not more than 20% of the captured species, the remaining 80% appearing only in the second eluate. In the case of 4 M urea, 1% CHAPS and 5% acetic acid, the elution power was augmented by a factor of three, up to 60%, whereas in the last case (8 M urea, 1% CHAPS and 5% acetic acid) the eluate represents about 80% (4 Â of eluant (i)) of the total [15] (i) 4 M urea, 1% CHAPS, 5% acetic acid; 320 proteins in PM; 332 immuno-depletion (ii) 6 M guanidine HCl, pH 6.0 [17] 4 M urea, 1% CHAPS, 5% acetic acid Many more SELDI peaks than control [18] (i) 8 M urea, 2% CHAPS, 5% acetic acid Identical spots in 2D maps (ii) 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris [19] 4 M urea, 1% CHAPS, 5% acetic acid Control: 157 spots; PM treated: 557 spots, IPGs pH 4-7 [20] 8 M urea, 2% CHAPS, 5% acetic acid Control: 48 peaks; PM-treated: 136 Peaks in SELDI [13] 4 M urea, 1% CHAPS Only high abundance proteins 1 mg to 40 mg/mL [21] 100 mM Tris-HCL pH 6.8, 4% SDS, 10% 2-ME, boil Untreated: 251 proteins; PM-treated: 693 proteins; saliva [14] 33.3% 2-propanol, 16.7% ACN, 0.1% TFA) 108 proteins unique to PM; 100 proteins unique to IgY; 404 total proteins; sera [23] (i) 8 M urea, 2% CHAPS, 5% acetic acid; Control: 211 proteins ; CPLL-treated: 512 a)…”

“…It turns out that there are essentially four main elution cocktails, which are listed here: (i) 4 M urea1 1% CHAPS [13]; (ii) 4 M urea11% CHAPS15% acetic acid [14,15,17,19]; (iii) 8 M urea12% CHAPS15% acetic acid [18,20,23]; and (iv) boiling 4% SDS125 mM DTT [21][22][23]. Briefly, the experimental protocol is as follows: 1 mL of serum is incubated for 3 h, on a rotating platform, with 50 mL of CPLL beads.…”