Differential Human Plasma Proteomics Based on AniBal Quantification and Peptide-level Off-Gel Isoelectric Focussing

2010 ◽  
Vol 3 ◽  
pp. PRI.S4851 ◽  
Author(s):  
Laure F. Marvin-Guy ◽  
Tatiana Zinger ◽  
Sandrine Wagnière ◽  
Véronique Parisod ◽  
Michael Affolter ◽  
...  
Keyword(s):  
Human Plasma ◽  
Plasma Proteins ◽  
Ion Trap ◽  
Quantitative Information ◽  
Proteome Coverage ◽  
Disease Biomarkers ◽  
Proteomics Approach ◽  
Peptide Level ◽  
Health And Disease ◽  
Human Plasma Proteome

Despite its enormous complexity, human plasma is still one of the most frequently used body fluids for identification and quantification of health and disease biomarkers. We have developed a new workflow for qualitative and quantitative analysis of human plasma proteins. The first step was to remove the seven most abundant plasma proteins (MARS). Moreover, in order to reduce the complexity of the sample and to increase protein and proteome coverage, Off-Gel fractionation was performed at peptide level. Our own stable isotope-based quantitative proteomics approach termed AniBAL was chosen for relative quantification of proteins between conditions. The method was developed with commercial human plasma and resulted in the identification of 85 proteins, of which 68 revealed quantitative information (Mascot database search combined with Peptide-/ProteinProphet validation). The combined methods consisting of MARS, AniBAL, Off-Gel and nano-LC-MS/MS on a Bruker HCT ion trap represent a new and efficient platform to quantify human plasma proteome differences between conditions. The method was also found technically compatible to a pair of human plasma pilot samples from the European FP6 project “DiOGenes”. Many of the identifiable/quantifiable proteins are relevant to obesity, diabetes and inflammation, which form the context of investigation within “DiOGenes”.

scholarly journals Undulating changes in human plasma proteome across lifespan are linked to disease

2019 ◽  
Author(s):  
Benoit Lehallier ◽  
David Gate ◽  
Nicholas Schaum ◽  
Tibor Nanasi ◽  
Song Eun Lee ◽  
...  
Keyword(s):  
Human Plasma ◽  
Plasma Proteins ◽  
Biological Pathways ◽  
Phenotypic Traits ◽  
Plasma Proteome ◽  
New Approach ◽  
Disease Biology ◽  
Age Related ◽  
Insight Into ◽  
Human Plasma Proteome

Aging is the predominant risk factor for numerous chronic diseases that limit healthspan. Mechanisms of aging are thus increasingly recognized as therapeutic targets. Blood from young mice reverses aspects of aging and disease across multiple tissues, pointing to the intriguing possibility that age-related molecular changes in blood can provide novel insight into disease biology. We measured 2,925 plasma proteins from 4,331 young adults to nonagenarians and developed a novel bioinformatics approach which uncovered profound non-linear alterations in the human plasma proteome with age. Waves of changes in the proteome in the fourth, seventh, and eighth decades of life reflected distinct biological pathways, and revealed differential associations with the genome and proteome of age-related diseases and phenotypic traits. This new approach to the study of aging led to the identification of unexpected signatures and pathways of aging and disease and offers potential pathways for aging interventions.

scholarly journals Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Zhiyun Cao ◽  
Sachin Yende ◽  
John A. Kellum ◽  
Renã A. S. Robinson
Keyword(s):  
Human Plasma ◽  
Exchange Chromatography ◽  
Differentially Expressed ◽  
Reference Database ◽  
Plasma Proteome ◽  
Proteome Coverage ◽  
Liquid Chromatography Tandem Mass ◽  
Dual Stage ◽  
Strong Cation Exchange Chromatography ◽  
Human Plasma Proteome

Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1) to provide increased depth in plasma proteome coverage and (2) to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS) column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC) were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%.

Extending the Depth of Human Plasma Proteome Coverage Using Simple Fractionation Techniques

2021 ◽  
Vol 20 (2) ◽  
pp. 1261-1279
Author(s):  
Gurjeet Kaur ◽  
Anne Poljak ◽  
Syed Azmal Ali ◽  
Ling Zhong ◽  
Mark J. Raftery ◽  
...  
Keyword(s):  
Human Plasma ◽  
Plasma Proteome ◽  
Proteome Coverage ◽  
Human Plasma Proteome

scholarly journals The Plasma Proteome

2021 ◽  
Author(s):  
Zhuo Zhen Chen ◽  
Wei-Cheih Wang ◽  
Lloyd Johnson ◽  
Jaimie Dufresne ◽  
Peter Bowden ◽  
...  
Keyword(s):  
Human Plasma ◽  
Ion Trap ◽  
Null Distribution ◽  
Linear Ion Trap ◽  
Plasma Proteome ◽  
Interconnected Network ◽  
Quaternary Amine ◽  
Edta Plasma ◽  
Human Plasma Proteome ◽  
Gene Symbols

Abstract INTODUCTIONThere is an urgent need for a simple and sensitive method to elucidate the human plasma proteome to find markers of disease, or therapeutic factors. Human plasma proteome may be obtained from tryptic peptides that results from native digestion using commonly available, sensitive and robust analytical instruments such as linear quadrupole, tandem mass spectrometers. METHODSThe human plasma proteome was elucidated from three independent human EDTA plasma populations analyzed by precipitation with acetonitrile (ACN) for quaternary amine (QA) micro-chromatography prior to native tryptic digestion for nano liquid chromatography, electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS results from authentic plasma and blank injection MS/MS noise controls were parsed into SQL Server along with the fit of the MS/MS spectra from the rigorous X!TANDEM for analysis with the R statistical system. A total of 13,408 gene symbols from tryptic (TRYP) and/or phosphor/tryptic (STYP) peptides showed ≥ 10 peptides with an FDR q ≤ 0.01 from fit of MS/MS spectra by X!TANDEM and were resolved from the null distribution of background noise showed a Chi Square value of χ2 ≥ 9 (p ≤ 0.005). RESULTSNative digestion of human EDTA plasma permitted the identification and quantification of ~ 13,408 protein gene symbols in plasma that showed low FDR (q≤0.01) from the fit of peptide MS/MS spectra and where observation frequency was resolved from the null distribution of random MS/MS spectra of source noise from recordings of blank injections. There was good agreement between the orbital ion trap (OIT) and the sensitive linear ion trap (LIT) as well as the tryptic versus phospho/tryptic peptides. A distinct subset of human cellular proteins showed a variety of specific interaction domains that formed a highly interconnected network in the plasma. DISCUSIONThe agreement between the fit of the peptide MS/MS spectra by the rigorous X!TANDEM algorithm versus random MS/MS spectra controls from blank noise injections demonstrated the reliability of the experimental approach. The highly interconnected network in the plasma confirmed that digestion of plasma under native conditions permitted the identification and quantification of the proteins in a population of human plasma samples. CONCLUSIONIt was feasible to identify more than ten thousand proteins from human plasma with high confidence using a simple linear ion trap after precipitation, quaternary amine chromatography, native digestion and nano spray analysis with a linear quadrupole ion trap.

scholarly journals Sphingosine 1-Phosphate Distribution in Human Plasma: Associations with Lipid Profiles

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Samar M. Hammad ◽  
Mohammed M. Al Gadban ◽  
Andrea J. Semler ◽  
Richard L. Klein
Keyword(s):  
Human Plasma ◽  
Plasma Proteins ◽  
Hdl Cholesterol ◽  
Physiological Significance ◽  
Plasma Lipoproteins ◽  
Total Plasma ◽  
Individual Subject ◽  
Sphingosine 1 Phosphate ◽  
Health And Disease ◽  
The Individual

The physiological significance of sphingosine 1-phosphate (S1P) transport in blood has been debated. We have recently reported a comprehensive sphingolipid profile in human plasma and lipoprotein particles (VLDL, LDL, and HDL) using HPLC-MS/MS (Hammad et al., 2010). We now determined the relative concentrations of sphingolipids including S1P in the plasma subfraction containing lipoproteins compared to those in the remaining plasma proteins. Sphingomyelin and ceramide were predominantly recovered in the lipoprotein-containing fraction. Total plasma S1P concentration was positively correlated with S1P concentration in the protein-containing fraction, but not with S1P concentration in the lipoprotein-containing fraction. The percentage of S1P transported in plasma lipoproteins was positively correlated with HDL cholesterol (HDL-C) concentration; however, S1P transport in lipoproteins was not limited by the concentration of HDL-C in the individual subject. Thus, different plasma pools of S1P may have different contributions to S1P signaling in health and disease.

Plasma Components which Interfere with Ristocetin-induced Platelet Aggregation

1975 ◽  
Vol 33 (03) ◽  
pp. 540-546 ◽  
Author(s):  
Robert F Baugh ◽  
James E Brown ◽  
Cecil Hougie
Keyword(s):  
Platelet Aggregation ◽  
Human Plasma ◽  
Plasma Proteins ◽  
Binding Protein ◽  
Plasma Heating ◽  
Protein S ◽  
Fold Increase ◽  
Normal Human Plasma ◽  
Preliminary Examination ◽  
Normal Human

SummaryNormal human plasma contains a component or components which interfere with ristocetin-induced platelet aggregation. Preliminary examination suggests a protein (or proteins) which binds ristocetin and competes more effectively for ristocetin than do the proteins involved in ristocetin-induced platelet aggregation. The presence of this protein in normal human plasma also prevents ristocetin-induced precipitation of plasma proteins at levels of ristocetin necessary to produce platelet aggregation (0.5–2.0 mg/ml). Serum contains an apparent two-fold increase of this component when compared with plasma. Heating serum at 56° for one hour results in an additional 2 to 4 fold increase. The presence of a ristocetin-binding protein in normal human plasma requires that this protein be saturated with ristocetin before ristocetin-induced platelet aggregation will occur. Variations in the ristocetin-binding protein(s) will cause apparent discrepancies in ristocetin-induced platelet aggregation in normal human plasmas.

Studies on the Fibrinolytic System in Human Plasma: Quantitative Determination of Plasminogen Activators and Proactivators

1979 ◽  
Vol 41 (02) ◽  
pp. 365-383 ◽  
Author(s):  
C Kluft
Keyword(s):  
Pilot Study ◽  
Plasma Level ◽  
Human Plasma ◽  
Plasminogen Activators ◽  
Venous Occlusion ◽  
Quantitative Information ◽  
Optimal Recovery ◽  
Dextran Sulphate ◽  
Baseline Levels

SummaryEffects due to plasma plasminogen activators and proactivators are usually studied in assay systems where inhibitors influence the activity and where the degree of activation of proactivators is unknown. Quantitative information on activator and proactivator levels in plasma is therefore not availableStudies on the precipitating and activating properties of dextran sulphate in euglobulin fractionation presented in this paper resulted in the preparation of a fraction in which there was optimal recovery and optimal activation of a number of plasminogen activators and proactivators from human plasma. The quantitative assay of these activators on plasminogen-rich fibrin plates required the addition of flufenamate to eliminate inhibitors. The response on the fibrin plates (lysed zones) could be coverted to arbitrary blood activator units (BAU). Consequently, a new activator assay which enables one to quantitatively determine the plasma level of plasminogen activators and proactivators together is introduced.Two different contributions could be distinguished: an activity originating from extrinsic activator and one originating from intrinsic proactivators. The former could be assayed separately by means of its resistance to inhibition by Cl-inactivator. Considering the relative concentrations of extrinsic and intrinsic activators, an impression of the pattern of activator content in plasma was gained. In morning plasma with baseline levels of fibrinolysis, the amount of extrinsic activator was negligible as compared to the level of potentially active intrinsic activators. Consequently, the new assay nearly exclusively determines the level of intrinsic activators in morning plasma. A pilot study gave a fairly stable level of 100 ± 15 BAU/ml (n = 50). When fibrinolysis was stimulated by venous occlusion (15 min), the amount of extrinsic activator was greatly increased, reaching a total activator level of 249 ± 27 BAU/ml (n = 7).

Detectability of Plasma Proteins in SRM Measurements

2018 ◽  
Vol 16 (1) ◽  
pp. 74-81 ◽  
Author(s):  
Olga I. Kiseleva ◽  
Elena A. Ponomarenko ◽  
Yulia A. Romashova ◽  
Ekaterina V. Poverennaya ◽  
Andrey V. Lisitsa
Keyword(s):  
Blood Plasma ◽  
Human Plasma ◽  
Limit Of Detection ◽  
Human Blood Plasma ◽  
Analytical Sensitivity ◽  
Plasma Proteome ◽  
Protein Targets ◽  
Blood Plasma Sample ◽  
Specificity And Sensitivity ◽  
Human Plasma Proteome

Background: Liquid chromatography coupled with targeted mass spectrometry underwent rapid technical evolution during last years and has become widely used technology in clinical laboratories. It offers confident specificity and sensitivity superior to those of traditional immunoassays. However, due to controversial reports on reproducibility of SRM measurements, the prospects of clinical appliance of the method are worth discussing. </P><P> Objective: The study was aimed at assessment of capabilities of SRM to achieve a thorough assembly of the human plasma proteome. </P><P> Method: We examined set of 19 human blood plasma samples to measure 100 proteins, including FDA-approved biomarkers, via SRM-assay. </P><P> Results: Out of 100 target proteins 43 proteins were confidently detected in at least two blood plasma sample runs, 36 and 21 proteins were either not detected in any run or inconsistently detected, respectively. Empiric dependences on protein detectability were derived to predict the number of biological samples required to detect with certainty a diagnostically relevant quantum of the human plasma proteome. </P><P> Conclusion: The number of samples exponentially increases with an increase in the number of protein targets, while proportionally decreasing to the logarithm of the limit of detection. Analytical sensitivity and enormous proteome heterogeneity are major bottlenecks of the human proteome exploration.

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