Biomarkers In Human Plasma Research Articles

Plasma proteomic study of acute mountain sickness susceptible and resistant individuals

Although extensive studies have focused on the development of acute mountain sickness (AMS), the exact mechanisms of AMS are still obscure. In this study, we used isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis to identify novel AMS−associated biomarkers in human plasma. After 9 hours of hypobaric hypoxia the abundance of proteins related to tricarboxylic acid (TCA) cycle, glycolysis, ribosome, and proteasome were significantly reduced in AMS resistant (AMS−) group, but not in AMS susceptible (AMS+) group. This suggested that AMS− individuals could reduce oxygen consumption via repressing TCA cycle and glycolysis, and reduce energy consumption through decreasing protein degradation and synthesis compared to AMS+ individuals after acute hypoxic exposure. The inflammatory response might be decreased resulting from the repressed TCA cycle. We propose that the ability for oxygen consumption reduction may play an important role in the development of AMS. Our present plasma proteomic study in plateau of the Han Chinese volunteers gives new data to address the development of AMS and potential AMS correlative biomarkers.

Sample preparation for detection of low abundance proteins in human plasma using ultra-high performance liquid chromatography coupled with highly accurate mass spectrometry.

Proteomics is a valuable approach to discover biomarkers in human plasma for early diagnosis. However, detection of biomarkers in the plasma is still challenging because of its large protein content. In our study, we established albumin/IgG depletion methods for identification of low abundance proteins using two commercial kits with additional buffer conditions and various concentrations of cold acetone. Trypsin digestion, desalting, and data-dependent acquisition were also optimized. More than 80% depletion of albumin/IgG was achieved with two commercial kits and 98% depletion of albumin was obtained with 70% cold acetone. Recovery of four reference proteins, BNP (47-76), insulin, cytochrome c, and ubiquitin was obtained in all optimized methods. The best recovery of reference proteins was obtained using the ProteoExtract albumin/IgG removal kit with buffer A (61%-106%). After cold acetone precipitation, three reference proteins were recovered more than 48% except ubiquitin (12%). The number of identified proteins by Mascot was 28, 35, 17, and 34 for ProteoExtract, ProteoPrep, 70%, and 50% cold acetone, respectively. Furthermore, optimized methods detected MS/MS fragmentation patterns of elevated BNP in patient samples with cardiac disease. Our study provides the conditions for efficient biomarker discovery by minimal removing of high abundant proteins.

In-Depth, Reproducible Analysis of Human Plasma Using IgY 14 and SuperMix Immunodepletion.

Identification of cancer and other disease biomarkers in human plasma has been exceptionally challenging due to the complex nature of plasma and the presence of a moderate number of high- and medium-abundance proteins which mask low-abundance proteins of interest. As a result, immunoaffinity depletion formats combining multiple antibodies to target the most abundant plasma proteins have become the first stage in most plasma proteome discovery schemes. This protocol describes the use of tandem IgY 14 and SuperMix immunoaffinity depletion to reproducibly remove >99% of total plasma protein. This greatly increases the depth of analysis of human plasma proteomes. Depleted plasma samples can then be analyzed in a single high-resolution LC-MS/MS run on a Q Exactive Plus mass spectrometer, followed by label-free quantitation. If greater depth of analysis is desired, the depleted plasma can be further fractionated by separating the sample for a short distance on a 1D SDS gel and cutting the gel into uniform slices prior to trypsin digestion. Alternatively, the depleted plasma can be reduced, alkylated, and digested with trypsin followed by high-pH reversed-phase HPLC separation.

Hybrid Synthetic Receptors on MOSFET Devices for Detection of Prostate Specific Antigen in Human Plasma.

The study reports the use of extended gate field-effect transistors (FET) for the label-free and sensitive detection of prostate cancer (PCa) biomarkers in human plasma. The approach integrates for the first time hybrid synthetic receptors comprising of highly selective aptamer-lined pockets (apta-MIP) with FETs for sensitive detection of prostate specific antigen (PSA) at clinically relevant concentrations. The hybrid synthetic receptors were constructed by immobilizing an aptamer-PSA complex on gold and subjecting it to 13 cycles of dopamine electropolymerization. The polymerization resulted in the creation of highly selective polymeric cavities that retained the ability to recognize PSA post removal of the protein. The hybrid synthetic receptors were subsequently used in an extended gate FET setup for electrochemical detection of PSA. The sensor was reported to have a limit of detection of 0.1 pg/mL with a linear detection range from 0.1 pg/mL to 1 ng/mL PSA. Detection of 1-10 pg/mL PSA was also achieved in diluted human plasma. The present apta-MIP sensor developed in conjunction with FET devices demonstrates the potential for clinical application of synthetic hybrid receptors for the detection of clinically relevant biomarkers in complex samples.

LC-MS/MS method for quantitation of seven biomarkers in human plasma for the assessment of insulin resistance and impaired glucose tolerance.

Early detection of insulin resistance (IR) and/or impaired glucose tolerance (IGT) is crucial for delaying and preventing the progression toward type 2 diabetes. We recently developed and validated a straightforward metabolite-based test for the assessment of IR and IGT in a single LC-MS/MS method. Plasma samples were diluted with isotopically-labeled internal standards and extracted by simple protein precipitation. The extracts were analyzed by LC-MS/MS for the quantitation of 2-hydroxybutyric acid (0.500-40.0μg/mL), 3-hydroxybutyric acid (1.00-80.0μg/mL), 4-methyl-2-oxopentanoic acid (0.500-20.0μg/mL), 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphocholine (2.50-100μg/mL), oleic acid (10.0-400μg/mL), pantothenic acid (0.0100-0.800μg/mL), and serine (2.50-100μg/mL). Liquid chromatography was carried out on a reversed phase column with a run time of 3.1min and the mass spectrometer operated in negative MRM mode. Method validation was performed on three identical LC-MS/MS systems with five runs each. Sufficient linearity (R2>0.99) was observed for all the analytes over the ranges. The imprecision (CVs) was found to be less than 5.5% for intra-run and less than 5.8% for inter-run for the seven analytes. The analytical recovery was determined to be between 96.3 and 103% for the seven analytes. This fast and robust method has subsequently been used for patient sample analysis for the assessment of IR and IGT.

Polyamine Metabolites Profiling for Characterization of Lung and Liver Cancer Using an LC-Tandem MS Method with Multiple Statistical Data Mining Strategies: Discovering Potential Cancer Biomarkers in Human Plasma and Urine

Polyamines, one of the most important kind of biomarkers in cancer research, were investigated in order to characterize different cancer types. An integrative approach which combined ultra-high performance liquid chromatography—tandem mass spectrometry detection and multiple statistical data processing strategies including outlier elimination, binary logistic regression analysis and cluster analysis had been developed to discover the characteristic biomarkers of lung and liver cancer. The concentrations of 14 polyamine metabolites in biosamples from lung (n = 50) and liver cancer patients (n = 50) were detected by a validated UHPLC-MS/MS method. Then the concentrations were converted into independent variables to characterize patients of lung and liver cancer by binary logic regression analysis. Significant independent variables were regarded as the potential biomarkers. Cluster analysis was engaged for further verifying. As a result, two values was discovered to identify lung and liver cancer, which were the product of the plasma concentration of putrescine and spermidine; and the ratio of the urine concentration of S-adenosyl-l-methionine and N-acetylspermidine. Results indicated that the established advanced method could be successfully applied to characterize lung and liver cancer, and may also enable a new way of discovering cancer biomarkers and characterizing other types of cancer.

Femtomolar Detection of Tau Proteins in Undiluted Plasma Using Surface Plasmon Resonance.

The ability to directly detect Tau protein and other neurodegenerative biomarkers in human plasma at clinically relevant concentrations continues to be a significant hurdle for the establishment of diagnostic tests for Alzheimer's disease (AD). In this article, we introduce a new DNA aptamer/antibody sandwich assay pairing and apply it for the detection of human Tau 381 in undiluted plasma at concentrations as low as 10 fM. This was achieved on a multichannel surface plasmon resonance (SPR) platform with the challenge of working in plasma overcome through the development of a tailored mixed monolayer surface chemistry. In addition, a robust methodology was developed involving various same chip control measurements on reference channels to which the detection signal was normalized. Comparative measurements in plasma between SPR and enzyme-linked immunosorbent assay (ELISA) measurements were also performed to highlight both the 1000-fold performance enhancement of SPR and the ability to measure both spiked and native concentrations that are not achievable with ELISA.

A Targeted Metabolomics MRM-MS Study on Identifying Potential Hypertension Biomarkers in Human Plasma and Evaluating Acupuncture Effects.

The critical role of metabolic abnormality in hypertension is increasingly recognized, but its biomarkers are not clearly identified. In this study, 47 chemical compounds recorded by literature were employed as target metabolites of essential hypertension (EH). We detected their content in the plasma of EH patients and healthy subjects by using the Multiple Reaction Monitoring-Mass Spectrometry (MRM-MS). After screening the most altered compounds, acupuncture was used to treat patients for 3 months and these plasma metabolites were tested again. The results showed that oleic acid (OA) and myoinositol (MI) were the most important differential metabolites between the hypertensive plasma and the healthy plasma. They were also closely correlated with 24-hour blood pressure and nocturnal dipping. Moreover, plasma OA and MI could be restored to normal levels by acupuncture, accompanying with reduction of 24-hour systolic and diastolic blood pressure [from 145.10 ± 9.28 mm Hg to 140.70 ± 9.59 mm Hg (P < 0.0001), and 88.35 ± 7.92 mm Hg to 85.86 ± 7.95 mm Hg (P = 0.0024), respectively] and improvement of circadian blood pressure rhythm. This study demonstrated that plasma OA and MI were potential hypertension biomarkers and they could be used to preliminarily assess the treating effects such as acupuncture.

A standardized kit for automated quantitative assessment of candidate protein biomarkers in human plasma.

An increasingly popular mass spectrometry-based quantitative approach for health-related research in the biomedical field involves the use of stable isotope-labeled standards (SIS) and multiple/selected reaction monitoring (MRM/SRM). To improve inter-laboratory precision and enable more widespread use of this 'absolute' quantitative technique in disease-biomarker assessment studies, methods must be standardized. Results/methodology: Using this MRM-with-SIS-peptide approach, we developed an automated method (encompassing sample preparation, processing and analysis) for quantifying 76 candidate protein markers (spanning >4 orders of magnitude in concentration) in neat human plasma. The assembled biomarker assessment kit - the 'BAK-76' - contains the essential materials (SIS mixes), methods (for acquisition and analysis), and tools (Qualis-SIS software) for performing biomarker discovery or verification studies in a rapid and standardized manner.

Population pharmacokinetic and pharmacodynamic analysis of plasma Aβ40 and Aβ42 following single oral doses of the BACE1 inhibitor AZD3839 to healthy volunteers.

Modulating deposition of Aβ-containing plaques in the brain may be beneficial in treating Alzheimer's disease. β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors have been shown to reduce Aβ in plasma and CSF in healthy volunteers. In this study safety, pharmacokinetics and pharmacodynamics that is reduction of the plasma biomarkers Aβ40 and Aβ42 , of the BACE1 inhibitor AZD3839 were evaluated. Single oral ascending doses (1-300 mg) of AZD3839 were administered to 54 young healthy volunteers in a randomized, double-blind, placebo-controlled study. The data was analyzed using non-linear mixed effects modeling. AZD3839 reduced Aβ40 and Aβ42 in plasma with estimated potencies (EC50 ) of 46 and 59 nM, respectively, and a maximum effect of approximately 55%. This was in excellent agreement with the concentration-response relationships obtained in mouse and guinea pig. AZD3839 exposure displayed non-linear kinetics, described by a three-compartment model with a saturated binding compartment and an increase in bioavailability with dose. AZD3839 was safe, although, a dose-dependent QTcF prolongation was observed (mean 20 milliseconds at 300 mg). In conclusion, AZD3839 reduced plasma Aβ40 and Aβ42 , demonstrating clinical peripheral proof of mechanism. Pre-clinical models were predictive for the effect of AZD3839 on the human plasma biomarker in a strictly quantitative manner.

A lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform for identification of multiple liver cancer biomarkers in human plasma

Aberrantly glycosylated proteins related to liver cancer progression were captured with specific lectin and identified from human plasma by multiple reaction monitoring (MRM) mass spectrometry as multiple biomarkers for hepatocellular carcinoma (HCC). The lectin fractionation for fucosylated protein glycoforms in human plasma was conducted with a fucose-specific aleuria aurantia lectin (AAL). Following tryptic digestion of the lectin-captured fraction, plasma samples from 30 control cases (including 10 healthy, 10 hepatitis B virus [HBV], and 10 cirrhosis cases) and 10 HCC cases were quantitatively analyzed by MRM to identify which glycoproteins are viable HCC biomarkers. A1AG1, AACT, A1AT, and CERU were found to be potent biomarkers to differentiate HCC plasma from control plasmas. The AUROC generated independently from these four biomarker candidates ranged from 0.73 to 0.92. However, the lectin-coupled MRM assay with multiple combinations of biomarker candidates is superior statistically to those generated from the individual candidates with AUROC more than 0.95, which can be an alternative to the immunoassay inevitably requiring tedious development of multiple antibodies against biomarker candidates to be verified. Eventually the lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform was found to be efficient to identify multiple biomarkers from human plasma according to cancer progression.

MRM‐based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma

A highly-multiplexed MRM-based assay for determination of cardiovascular disease (CVD) status and disease classification has been developed for clinical research. A high-flow system using ultra-high performance LC and an Agilent 6490 triple quadrupole mass spectrometer, equipped with an ion funnel, provided ease of use and increased the robustness of the assay. The assay uses 135 stable isotope-labeled peptide standards for the quantitation of 67 putative biomarkers of CVD in tryptic digests of whole plasma in a 30-min assay. Eighty-five analyses of the same sample showed no loss of sensitivity (<20% CV for 134/135 peptides) and no loss of retention time accuracy (<0.5% CV for all peptides). The maximum linear dynamic range of the MRM assays ranged from 10(3) -10(5) for 106 of the assays. Excellent linear responses (r >0.98) were obtained for 117 of the 135 peptide targets with attomole level limits of quantitation (<20% CV and accuracy 80-120%) for 81 of the 135 peptides. The assay presented in this study is easy to use, robust, sensitive, and has high-throughput capabilities through short analysis time and complete automated sample preparation. It is therefore well suited for CVD biomarker validation and discovery in plasma.

Development of a Chip/Chip/SRM Platform Using Digital Chip Isoelectric Focusing and LC-Chip Mass Spectrometry for Enrichment and Quantitation of Low Abundance Protein Biomarkers in Human Plasma

Protein biomarkers are critical for diagnosis, prognosis, and treatment of disease. The transition from protein biomarker discovery to verification can be a rate limiting step in clinical development of new diagnostics. Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) is becoming an important tool for biomarker verification studies in highly complex biological samples. Analyte enrichment or sample fractionation is often necessary to reduce sample complexity and improve sensitivity of SRM for quantitation of clinically relevant biomarker candidates present at the low ng/mL range in blood. In this paper, we describe an alternative method for sample preparation for LC-SRM MS, which does not rely on availability of antibodies. This new platform is based on selective enrichment of proteotypic peptides from complex biological peptide mixtures via isoelectric focusing (IEF) on a digital ProteomeChip (dPC) for SRM quantitation using a triple quadrupole (QQQ) instrument with an LC-Chip (Chip/Chip/SRM). To demonstrate the value of this approach, the optimization of the Chip/Chip/SRM platform was performed using prostate specific antigen (PSA) added to female plasma as a model system. The combination of immunodepletion of albumin and IgG with peptide fractionation on the dPC, followed by SRM analysis, resulted in a limit of quantitation of PSA added to female plasma at the level of ∼1-2.5 ng/mL with a CV of ∼13%. The optimized platform was applied to measure levels of PSA in plasma of a small cohort of male patients with prostate cancer (PCa) and healthy matched controls with concentrations ranging from 1.5 to 25 ng/mL. A good correlation (r(2) = 0.9459) was observed between standard clinical ELISA tests and the SRM-based assay. Our data demonstrate that the combination of IEF on the dPC and SRM (Chip/Chip/SRM) can be successfully applied for verification of low abundance protein biomarkers in complex samples.

Targeted Mass Spectrometric Approach for Biomarker Discovery and Validation with Nonglycosylated Tryptic Peptides from N-linked Glycoproteins in Human Plasma

A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or expensive antibody preparations.

Human urine proteomics: building a list of human urine cancer biomarkers

In the last decade, several reports have focused on the identification and characterization of proteins present in urine. In an effort to build a list of proteins of interest as biomarkers, we reviewed the largest urine proteomes and built two updated lists of proteins of interest (available as supplementary tables). The first table includes a consensus list of 443 proteins found in urine by independent laboratories and reported on the top three largest urine proteomes currently published. This consensus list of proteins could serve as biomarkers to diagnose, monitor and manage a number of diseases. Here, we focus on a reduced list of 35 proteins with potential interest as cancer biomarkers in urine following two criteria: first, proteins previously detected in urine using bottom-up proteomic experiments, and second, those suggested as cancer protein biomarkers in human plasma. In an effort to standardize the information presented and its use in future studies, here we include the updated International Protein Index (v. 3.80) and primary Swiss-Prot accession numbers, official gene symbols and recommended full names. The main variables that influence urine proteomic experiments are also discussed.

Solid-phase hexapeptide ligand libraries open up new perspectives in the discovery of biomarkers in human plasma

Background Pre-treatment of plasma with hexapeptide ligand libraries prior to proteomic analysis is well documented. However, the maintenance of biomarker abundance throughout the different pre-analytical steps is required for a potential application of differential proteomics in clinical studies. Methods We combined the use of an amino-terminal hexapeptide ligand library and its carboxyl-terminal version with a sequential elution strategy of the proteins/peptides bound to the beads, followed by either mass spectrometry or 2D electrophoresis analyses. Results We show the maintenance of C-reactive protein abundance (a marker of inflammation) throughout the process (including hexapeptide bead treatment and proteomic analysis) in patients presenting high and low levels of this protein. In parallel, we assessed the contribution of this workflow to increasing the number of potential biomarkers detected and its suitability for a clinical study on approximately a hundred samples, as well as the reproducibility of the process. Conclusions Pre-treatment with hexapeptide ligand librairies opens up new perspectives in the discovery of biomarkers in human plasma by improving the detection of new species while maintaining their original differential abundance. This approach is also suitable for an application in a clinical proteomic study of at least 100 samples.

Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple- Reaction–Monitoring Mass Spectrometry

If liquid-chromatography-multiple-reaction-monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay - 36.6; S(x|y) = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; S(x|y) = 7.9 for apoB). Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability.

High‐abundance protein depletion: Comparison of methods for human plasma biomarker discovery

This article was originally published in Electrophoresis 2010, 31, 471–482, DOI 10.1002/elps.200900286

High‐abundance protein depletion: Comparison of methods for human plasma biomarker discovery

Affinity depletion of abundant proteins from human plasma has become a routine sample preparation strategy in proteomics used prior to protein identification and quantitation. To date, there have been limited published studies comparing the performance of commercially available depletion products. We conducted a thorough evaluation of six depletion columns using 2-DE combined with sophisticated image analysis software, examining the following criteria: (i) efficiency of high-abundance protein depletion, (ii) non-specific removal of other than the targeted proteins and (iii) total number of protein spots detected on the gels following depletion. From all the products investigated, the Seppro IgY system provided the best results. It displayed the greatest number of protein spots on the depleted plasma gels, minimal non-specific binding and high efficiency of abundant protein removal. Nevertheless, the increase in the number of detected spots compared with the second best performing and cheaper multiple affinity removal column (MARC) was not shown to be statistically significant. The ProteoPrep spin column, considered to be the "deepest" depletion technique available at the time of conducting the study, surprisingly displayed significantly fewer spots on the flow-through fraction gels compared with both the Seppro and the MARC. The spin column format and low plasma capacity were also found to be impractical for 2-DE. To conclude, we succeeded in providing an overview of the depletion columns performances with regard to the three examined areas. Our study will serve as a reference to other scientists when deciding on the optimal product for their particular projects.

Analysis of angiotensin II receptor antagonist and protein markers at microliter level plasma by LC–MS/MS

An analytical method based on a green approach is proposed for clinical analysis. The proposed procedure involves the reduction of the sample preparation steps, the amounts of reagents and organic solvents. This simple and sensitive method for the analysis of clinical drug and biomarkers in human plasma was developed using LC connected to tandem mass spectrometry (LC–MS/MS) with a nanospray ion source. In this study, the desired drug and proteins were separated on a 5 and 10 cm RP C18 nano-flow column. Undesired polar substances in human plasma were washed out by using ACN:1% FA = 20:80 (v/v) as the loading mobile phase for drug analysis and good linearity was attainable. Only a small volume of human plasma (10 μL) was utilized for the monitoring of drug plasma concentration and significant proteins under clinical studies. All the sample preparation procedures and the analytical scheme were at microliter level. This strategy would lower the consumption of reagents and organic solvents and make a contribution toward the goal of reduction of pollution from analytical methods in general.