A Recombinant Protein Biomarker DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

AbstractCredible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). Here, we employed a mixture of recombinant proteins in DDA libraries to subsequently detect cancer-associated low abundance plasma proteins using SWATH/DIA. The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 human recombinant proteins that had been previously implicated as possible cancer biomarkers in both our own and other studies. The rPSL was then used to identify proteins from non-depleted colorectal cancer (CRC) plasmas by SWATH-MS. Most (32/36) of the proteins in the rPSL were reliably identified in plasma samples, including 8 proteins (BTC, CXCL10, IL1B, IL6, ITGB6, TGFα, TNF, TP53) not previously detected using high-stringency MS in human plasmas according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and post-digestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 were identified using DDA-MS. The 20 additional proteins exclusively identified by using the rPSL approach with SWATH were mostly lower abundance (i.e., <10ng/ml) plasma proteins. To mitigate FDR concerns, and replicating a more typical approach, the DDA rPSL was also merged into a human plasma DDA library. When SWATH identification was repeated using this merged library, the majority (33/36) of low abundance plasma proteins from the rPSL could still be identified using high-stringency HPP Guidelines v3.0 protein inference criteria.

Download Full-text

Multijunction Capillary Isoelectric Focusing Device Combined with Online Membrane-Assisted Buffer Exchanger Enables Isoelectric Point Fractionation of Intact Human Plasma Proteins for Biomarker Discovery

Analytical Chemistry ◽

10.1021/acs.analchem.5b03344 ◽

2015 ◽

Vol 87 (23) ◽

pp. 11840-11846 ◽

Cited By ~ 2

Author(s):

Mohammad Pirmoradian ◽

Juan Astorga-Wells ◽

Roman A. Zubarev

Keyword(s):

Human Plasma ◽

Isoelectric Focusing ◽

Isoelectric Point ◽

Plasma Proteins ◽

Biomarker Discovery ◽

Capillary Isoelectric Focusing

Download Full-text

Identification of Abnormal Proteins in Plasma from Gout Patients by LC-MS/MS

Separations ◽

10.3390/separations8060085 ◽

2021 ◽

Vol 8 (6) ◽

pp. 85

Author(s):

Lijin Shen ◽

Hanyang Dong ◽

Zhenchang Guo ◽

Guijin Zhai ◽

Kai Zhang

Keyword(s):

Human Plasma ◽

Plasma Proteins ◽

High Performance ◽

Clinical Analysis ◽

Kidney Damage ◽

Human Blood Plasma ◽

Protein Levels ◽

Differential Proteins ◽

High Level ◽

Abundance Proteins

A high level of uric acid may cause hyperuricemia, which further develops into gout, eventually leading to chronic kidney disease. However, the pathogenic mechanism remains largely unknown. To investigate the cause and block the transformation of hyperuricemia to related diseases, it is important to discover the alterations in protein levels between gout patients and non-gout individuals. To date, human blood plasma is still the predominant matrices for clinical analysis. Due to the high abundance, the proteins of plasma samples have strong shielding effects on low abundance proteins, thus, the information on low abundance protein expression is always masked, while the low abundance proteins of human plasma are often of great significance for the diagnosis and treatment of diseases. Therefore, it is very important to separate and analyze the plasma proteins. High-performance liquid chromatography (LC) tandem mass spectrometry (MS)-based proteomics has been developed as a powerful tool to investigate changes in the human plasma proteome. Here, we used LC-MS/MS to detect the differential proteins in the plasmas from simple gout patients, gout with kidney damage patients, and non-gout individuals. We identified 32 obviously differential proteins between non-gout and gout subjects and 10 differential proteins between simple gout and gout with kidney damage patients. These differential proteins were further analyzed to characterize their localization and functions. Additionally, the correlation analysis showed multiple relationships between the abnormal plasma proteins and clinical biochemical indexes, particularly for the immune-inflammatory response proteins. Furthermore, inflammation factors gelsolin (GSN) were confirmed. Our results offer a view of plasma proteins for studying biomarkers of gout patients.

Download Full-text

A Proteomic Approach for Plasma Biomarker Discovery with iTRAQ Labelling and OFFGEL Fractionation

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/927917 ◽

2010 ◽

Vol 2010 ◽

pp. 1-8 ◽

Cited By ~ 55

Author(s):

Emilie Ernoult ◽

Anthony Bourreau ◽

Erick Gamelin ◽

Catherine Guette

Keyword(s):

Plasma Proteins ◽

Biomarker Discovery ◽

Dynamic Range ◽

Human Blood Plasma ◽

Wide Dynamic Range ◽

Proteome Coverage ◽

Over Expression ◽

Proteomic Approach ◽

Human Proteins ◽

Proteome Profile

Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma “signatures,” which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 μg of plasma proteins.

Download Full-text

Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics

10.1101/472662 ◽

2018 ◽

Author(s):

Fredrik Edfors ◽

Björn Forsström ◽

Helian Vunk ◽

David Kotol ◽

Claudia Fredolini ◽

...

Keyword(s):

Recombinant Protein ◽

Human Plasma ◽

Recombinant Proteins ◽

Time Course ◽

Targeted Proteomics ◽

Protein Coding ◽

Protein Fragments ◽

Peptide Formation ◽

Digestion Kinetics ◽

Different Levels

AbstractThe availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labelled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labelled recombinant proteins prior to trypsin digestion enables short digestion protocols (≤60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINA5 and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.

Download Full-text

Plasma Components which Interfere with Ristocetin-induced Platelet Aggregation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647847 ◽

1975 ◽

Vol 33 (03) ◽

pp. 540-546 ◽

Cited By ~ 3

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.

Download Full-text

Validation of Simvastatin Analysis Methods in Human Blood Plasma (In Vitro) Using HPLC-UV Detector

International Journal of Drug Delivery Technology ◽

10.25258/ijddt.v8i2.13873 ◽

2018 ◽

Vol 8 (2) ◽

Author(s):

Iyan Sopyan ◽

Cynthia Jaya ◽

Driyanti Rahayu

Keyword(s):

Blood Plasma ◽

Human Plasma ◽

Disease Risk ◽

Protein Precipitation ◽

Precipitation Method ◽

Human Blood Plasma ◽

Analysis Method ◽

Uv Detector ◽

Number Of Patients ◽

System Suitability

The use of simvastatin (SV) increases along with the increasing number of patients with hyperlipidemia and cardiovascular disease risk factors. Consequently, this condition leads to the increasing need of analytical determination of SV in blood plasma. Analysis of SV in human plasma using protein precipitation method and HPLC with UV detector has not been reported. This research was purpose to find out the rapid, accurate, and valid of SV analysis method in human plasma. In this research plasma samples were treated with protein precipitation method. The analyte was then analyzed using HPLC with C18 column 250x4 mm and 5 µm of particle size, the mobile phase contained of phosphate buffer 0.01 M (pH 4.0) and acetonitrile 30:70 v/v with flow rate 1 mL/minute, and detected at 239 nm. The analysis method was validated based on some parameters, such as selectivity, accuracy, precision, repeatability, linearity, LOD, LOQ, and system suitability. The result showed selectivity represented by Rs was 2.870, repeatability by its CV less than 2%, and linearity by its coefficient correlation (r) 0.9992 for concentration range 0.08-0.32 ppm. Based on chromatogram peak area, LOD and LOQ were 0.0132 and 0.0440 ppm respectively, accuracy and precision were 86.25-89.36% and 0.66-1.81% were obtained. The result of system suitability test from retention time and chromatogram peak area showed by its CV less than 2%. The analysis method was proved to be valid for SV analysis in human plasma

Download Full-text

Detectability of Plasma Proteins in SRM Measurements

Current Proteomics ◽

10.2174/1570164615666180718151135 ◽

2018 ◽

Vol 16 (1) ◽

pp. 74-81 ◽

Cited By ~ 1

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. Objective: The study was aimed at assessment of capabilities of SRM to achieve a thorough assembly of the human plasma proteome. Method: We examined set of 19 human blood plasma samples to measure 100 proteins, including FDA-approved biomarkers, via SRM-assay. 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. 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.

Download Full-text

Glycosylation of Cancer Extracellular Vesicles: Capture Strategies, Functional Roles and Potential Clinical Applications

Cells ◽

10.3390/cells10010109 ◽

2021 ◽

Vol 10 (1) ◽

pp. 109

Author(s):

Álvaro M. Martins ◽

Cátia C. Ramos ◽

Daniela Freitas ◽

Celso A. Reis

Keyword(s):

Extracellular Vesicles ◽

Biomarker Discovery ◽

De Novo ◽

Recipient Cell ◽

Cell Communication ◽

Cancer Biomarkers ◽

Functional Roles ◽

Molecular Information ◽

Glycosylation Pathway ◽

Organ Tropism

Glycans are major constituents of extracellular vesicles (EVs). Alterations in the glycosylation pathway are a common feature of cancer cells, which gives rise to de novo or increased synthesis of particular glycans. Therefore, glycans and glycoproteins have been widely used in the clinic as both stratification and prognosis cancer biomarkers. Interestingly, several of the known tumor-associated glycans have already been identified in cancer EVs, highlighting EV glycosylation as a potential source of circulating cancer biomarkers. These particles are crucial vehicles of cell–cell communication, being able to transfer molecular information and to modulate the recipient cell behavior. The presence of particular glycoconjugates has been described to be important for EV protein sorting, uptake and organ-tropism. Furthermore, specific EV glycans or glycoproteins have been described to be able to distinguish tumor EVs from benign EVs. In this review, the application of EV glycosylation in the development of novel EV detection and capture methodologies is discussed. In addition, we highlight the potential of EV glycosylation in the clinical setting for both cancer biomarker discovery and EV therapeutic delivery strategies.

Download Full-text