Single Chain Variable Fragment Displaying M13 Phage Library Functionalized Magnetic Microsphere-Based Protein Equalizer for Human Serum Protein Analysis

Abstract

Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@ M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (GlyHCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC−ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@ M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification. S earching indicators or biomarkers for diseases is one of the most important issues in clinical proteomics. Among various body fluids, serum is the one of the most favorite clinical samples, because not only it contains most possible biomarkers of diseases, 1,2 but also the collection is minimally invasive.However, the enormous dynamic range of protein concentration makes serum the most difficult specimen to be dealt with by existing techniques. The few dozen of highabundance proteins make up 99% of overall contents, which seriously undermine the identification of low-abundance ones. 3−5 Therefore, the decrease of the dynamic range of protein concentration in serum is essential to discover lowabundance ones. Until now, there are two main strategies to decrease the dynamic range of protein concentration in serum, depletion of high-abundance proteins, 6−14 and equalization of protein abundance. 15−28 For depletion of high-abundance proteins in serum samples, several kinds of immnoaffinity columns have been widely used. 6−13 Recently, Zeng et al. 10 applied CaptureSelect depletion resins (from BAC. B.V.) to remove albumin and immunoglobulin from human serum, followed by highperformance multilectin affinity chromatography fractionation, isoelectric focusing fractionation, in-gel digestion, and reversedplase liquid chromatography−electrospray ionization-tandem mass sp...

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 2011–2014

“…The immobilization of a single chain variable fragment (scFv) displaying M13 phage library on magnetic microspheres for use as a protein equalizer for the treatment of human serum has been recently reported [27]. Firstly, the diluted human serum was loaded on 100 mg scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM).…”

Bacteriophage Capsid Modification by Genetic and Chemical Methods

“…Glutaraldehyde is a dialdehyde that is frequently used as a cross-linking agent to form chemically and thermally stable cross-links of biological materials. , Glutaraldehyde is present in many forms in solution leading to the precise mechanism and primary reactive species not being fully understood or agreed upon in the literature . Glutaraldehyde can react with several nucleophilic functional groups present in proteins including amines, thiols, phenols, and imidazole, but the ε-amino group of lysine was found to be the most reactive. , This method has been used to facilitate cross-linking phages to amine-functionalized magnetic microspheres and gold surfaces. , N-Terminal serine and threonine residues contain a β-amino alcohol motif that can specifically undergo oxidative cleavage by sodium periodate to generate an aldehyde handle . This handle can be subjected to a second reaction to add the desired conjugate.…”

“…262,263 This method has been used to facilitate cross-linking phages to amine-functionalized magnetic microspheres and gold surfaces. 110,264 N-Terminal serine and threonine residues contain a β-amino alcohol motif that can specifically undergo oxidative cleavage by sodium periodate to generate an aldehyde handle. 265 This handle can be subjected to a second reaction to add the desired conjugate.…”

Virus Outbreaks in Chemical and Biological Sensors

“…Filamentous phages have been immobilized on magnetic microspheres for PHAIA [62], serum protein analysis [92], and affinity separation [93]. Recently, Jeon et al immobilized fd virions on Au-coated microspheres with orientation-controlled manner, providing exceptionally large surface to volume ratio for the immobilization of capture antibodies in high density [91] (Figure 9).…”