Depletion and fractionation technologies in plasma proteomic analysis

Tam, Sun W.; Pirro, John P.; Hinerfeld, Douglas

doi:10.1586/14789450.1.4.411

Cited by 36 publications

(20 citation statements)

References 31 publications

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“…Since, human plasma can be easily obtained, several laboratories have developed various proteomic strategies for the purpose of using plasma proteins as a source for possible disease-related biomarkers [7][8][9][10][11][12][13][14][15]22]. However, the great variety of protein types present, as well as the extended dynamic range of protein concentrations greatly hinders the identification of plasma biomarkers.…”

Section: Discussionmentioning

confidence: 99%

Proteomic Analysis of Human Plasma Proteins by Two-Dimensional Gel Electrophoresis and by Antibody Arrays Following Depletion of High-Abundance Proteins

Desrosiers

Beaulieu

Buchanan

et al. 2007

Cell Biochem Biophys

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Detecting proteins that are present at lower levels in human plasma, for the identification of potential disease biomarkers, is complicated by a few highly abundant proteins. One promising strategy is the removal of these abundant proteins interfering with the analysis of plasma content by proteomic techniques. This study compared three affinity-based methods to remove the most abundant proteins in human plasma. Two of them, based on antibodies, which depletes the six or the 12 most abundant proteins, demonstrated the highest efficiency in enriching less abundant plasma proteins. Comparison of two anticoagulant treatments for plasma preparation, EDTA and CTAD, showed that this treatment influenced the patterns of lower-abundance proteins visible on 2-dimensional (2-D) gels. Several staining procedures including two fluorescent dyes, Sypro Ruby and Deep Purple, were also compared with a very sensitive silver staining method for the visualization of lower-abundance proteins on 2-D gels. Furthermore, treatments of lower-abundance plasma proteins with hydroxyethyl disulfide enhanced protein sharpness and resolution. The purpose of all these systematic comparisons was to select the most reliable methods in different steps of plasma preparation and handling as well as in analysis of proteins by 2-D gels to obtain highly reproducible patterns of lower-abundance plasma proteins. Importantly, the lower-abundance plasma proteins enriched by these optimized conditions were further analyzed by antibody microarrays allowing the identification of 61 proteins using 350 antibodies directed against signalling proteins suggesting that this proteomic strategy is a valuable approach for detecting potential plasma biomarkers.

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Section: Discussionmentioning

confidence: 99%

Proteomic Analysis of Human Plasma Proteins by Two-Dimensional Gel Electrophoresis and by Antibody Arrays Following Depletion of High-Abundance Proteins

Desrosiers

Beaulieu

Buchanan

et al. 2007

Cell Biochem Biophys

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“…2). After focusing, the region below pH 4.8, which did not contain proteins, was excised, and the 12 cm trimmed IPG strip was separated in second dimension using gels of four different heights (8,12,15, and 19 cm). The numbers of spots detected in these gels were 1039 l 36, 1304 l 40, 1312 l 38, and 1327 l 40, respectively.…”

Section: Resultsmentioning

confidence: 99%

Effect of separation dimensions on resolution and throughput using very narrow‐range IEF for 2‐DE after solution phase isoelectric fractionation of a complex proteome

Lee¹,

2009

J of Separation Science

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This paper describes how the lengths of the first and second dimensions in narrow pH-range 2-DE affect the number of detected protein spots, by analysis of human breast carcinoma cell line lysates prefractionated by solution phase IEF. The aim is to maximize throughput while minimizing experimental costs. In this study, systematic evaluation of narrow-range IPG strip lengths showed that separation distances were very important, with dramatic increases in resolution when longer gels were used. Compared with 7 cm minigels, maximal resolution was obtained using 18 and 24 cm IPG strips. Systematic evaluation of SDS-PAGE gel length showed a far weaker influence of separation length on resolution in the second dimension compared with that observed for the IEF dimension. There was little benefit in using separation distances greater than 12-15 cm, at least with currently available electrophoresis units. The work shows that regions of the IPG strip not containing any proteins can be excised to fit a smaller gel if prefractionation using IEF in solution has been performed. As expected, larger 2-DE gel volumes resulting from the use of longer IPG strips and second dimension gels decreased detection sensitivity when equal protein loads were used. However, this effect could be readily eliminated by increasing the loads applied to IPG strips.

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“…Several types of columns containing IgGs from different mammalian species (goat, mouse, rabbit, etc.) are commercially available [42]. Hinerfeld et al reported that chicken anti-human HSA immunoglobulin Y (IgY) antibodies -bound to hydrazide beads -featured high affinity towards human albumin, thus provided highly specific depletion [43].…”

Section: Affinity-based Depletion Of the High Abundant Plasma Proteinsmentioning

confidence: 99%

Medicinal Chemistry Meets Proteomics: Fractionation of the Human Plasma Proteome

Kovacs¹,

Guttman²

2013

Current Medicinal Chemistry

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Human plasma and its fractions/derivatives are frequently used materials in biomedicine as it contains thousands and thousands of proteins representing the majority of human proteome. Several important methods were developed in the past for the fractionation of this important biological fluid and their use for medicinal purposes. One of the greatest challenges is the very large dynamic range of plasma proteins ranging up to 10-12 orders of magnitude. Early attempts were mainly based on methods such as salting out or cold ethanol precipitation, as well as chromatography utilizing affinity, size exclusion, ion exchange and hydrophobic interaction techniques. More recently, fractionation applications started with the depletion of the high abundant plasma components, such as serum albumin and immunoglobulins, before isolating lower abundant proteins of interest. Plasma volumes were utilized from the milliliter scale for diagnostic applications to hundreds of liters for industrial scale plasma fractionation (e.g., medicinal product manufacturing). In this paper we review this important part of medicinal chemistry, highlighting the traditional methods along with some of their variations as well as the most significant recent achievements of the field.

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Depletion and fractionation technologies in plasma proteomic analysis

Cited by 36 publications

References 31 publications

Proteomic Analysis of Human Plasma Proteins by Two-Dimensional Gel Electrophoresis and by Antibody Arrays Following Depletion of High-Abundance Proteins

Proteomic Analysis of Human Plasma Proteins by Two-Dimensional Gel Electrophoresis and by Antibody Arrays Following Depletion of High-Abundance Proteins

Effect of separation dimensions on resolution and throughput using very narrow‐range IEF for 2‐DE after solution phase isoelectric fractionation of a complex proteome

Medicinal Chemistry Meets Proteomics: Fractionation of the Human Plasma Proteome

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