Biomedical subjects

Dobrin Nedelkov

Publications and source records attributed to Dobrin Nedelkov.

At least 19 recordsLinked to original sources

PubMed2006

Surface plasmon resonance imaging measurements of antibody arrays for the multiplexed detection of low molecular weight protein biomarkers.

This paper describes a simple methodology for the creation of high-density multiplexed antibody arrays on gold surfaces that can be used to detect low molecular weight protein biomarkers with surface plasmon resonance imaging (SPRI). A one-step carbonyldiimidazole (CDI) surface reaction was utilized to attach antibodies onto alkanethiol-modified gold surfaces and characterized with polarization modulation FT-IR reflection absorption spectroscopy. The CDI chemistry was then employed to create an antibody microarray with array element sizes varying from 750 microm down to 200 microm. As a demonstration, a three-component antibody array was employed to detect two clinically important protein biomarkers, beta2-microglobulin (11.8 kDa) and cystatin C (13.4 kDa). SPRI measurements could simultaneously detect both of these small unlabeled proteins with no cross talk at solution concentrations from 300 nM down to 1 nM. In addition, the adsorption strengths of these biomarkers onto an antibody array were measured with SPRI and compared to those obtained from the kinetic analysis of single-channel angle shift SPR measurements.

Antibodies↗

PubMed2006

Population proteomics: the concept, attributes, and potential for cancer biomarker research.

This review outlines the concept of population proteomics and its implication in the discovery and validation of cancer-specific protein modulations. Population proteomics is an applied subdiscipline of proteomics engaging in the investigation of human proteins across and within populations to define and better understand protein diversity. Population proteomics focuses on interrogation of specific proteins from large number of individuals, utilizing top-down, targeted affinity mass spectrometry approaches to probe protein modifications. Deglycosylation, sequence truncations, side-chain residue modifications, and other modifications have been reported for myriad of proteins, yet little is know about their incidence rate in the general population. Such information can be gathered via population proteomics and would greatly aid the biomarker discovery efforts. Discovery of novel protein modifications is also expected from such large scale population proteomics, expanding the protein knowledge database. In regard to cancer protein biomarkers, their validation via population proteomics-based approaches is advantageous as mass spectrometry detection is used both in the discovery and validation process, which is essential for the detection of those structurally modified protein biomarkers.

Biomarkers, Tumor↗

PubMed2006

Development of recombinant-based mass spectrometric immunoassay with application to resistin expression profiling.

This report addresses the need for additional assays for human resistin (hRES) by developing a rational progression of the mass spectrometric immunoassay to incorporate recombinant proteins. The recombinant-based hRES mass spectrometric immunoassay (RES-MSIA) was initially developed for the qualitative analysis of the human resistin homodimer from normal (healthy) plasma samples. The method involved selective extraction and detection of both endogenous and recombinant resistant proteins. RES-MSIA was then applied to the rigorous quantification of resistin. The resistin standard addition curve was constructed from serially diluted concentrations of rhRES using endogenous hRES, inherent in the human plasma, as the internal reference standard (IRS). The roles of endogenous and recombinant resistin were subsequently reversed, using rhRES as the IRS during RES-MSIA quantification. Concurrently, the relative ratio of hRES to rhRES was used as an ancillary technique to rapidly determine the relative concentration of hRES in each of plasma samples. Overall, normal hRES levels determined by RES-MSIA were found to be comparable to those selected and determined by ELISA. With regard to gender, female donor samples were slightly elevated over males. Four single cardiac samples were analyzed and found to have hRES concentrations approximately three times that of the normal. The recombinant-based RES-MSIA is rapid and is amendable to parallel high-throughput robotic processing of resistin related disease cohorts.

Gene Expression Profiling↗

PubMed2006

Surface plasmon resonance-enabled mass spectrometry arrays.

Biosensors that utilize surface plasmon resonance (SPR) as a method of detection of protein interactions can be used for selective separation of proteins prior to MS analysis. The combination of SPR and MS results in a unique multiplexed detection technology capable of both quantitative and qualitative protein analysis. To further the development of a high-throughput SPR-MS approach, the possibility of arraying binding ligands on SPR chips for affinity capture of proteins and their MS analysis was explored. Antibodies to beta-2-microglobulin, cystatin C, transferrin, and insulin-like growth factors I and II were arrayed on a large number of SPR chips. Human plasma samples were injected over the antibody array chips in an SPR Biosensor, after which on-chip MS analysis was performed to detect the bound proteins. Signals from the targeted proteins were observed for each antibody-derivatized chip, indicating successful antibody immobilization and protein capture. The SPR-MS arrays are robust, highly reproducible, and are capable of high-throughput analysis.

Antibodies↗

PubMed2006

Multiplexed mass spectrometric immunoassay in biomarker research: a novel approach to the determination of a myocardial infarct.

Reported here is the development of a multiplexed mass spectrometric immunoassay (MSIA) for the detection of myocardial infarction (MI). The assay is the product of a study that systematically progresses from biomarker discovery--to identification and verification--to assay design, data analysis, and statistical challenge. During targeted population proteomics investigations, two novel biomarkers, serum amyloid A1alpha and S-sulfated transthyretin, were found to be responsive to MI. These putative markers were subsequently screened in larger cohorts of individuals to verify their responsiveness toward MI. Upon verification, a multiplexed assay was designed that was capable of simultaneously monitoring the new markers plus a previously established MI-marker (myoglobin). The multiplexed MSIA was applied to two 96-sample sets comprised of 48-MI/48-healthy and 19-MI/77-healthy, which served as training and case cohorts, respectively. Data evaluation using either preset reference levels or multivariate analysis exhibited sensitivities and specificities of >97%. These findings illustrate the importance of using systematic approaches in clinical proteomics to discover biomarkers and produce high-performance assays relevant to disease.

Biomarkers↗

PubMed2006

Quantitative multiplexed C-reactive protein mass spectrometric immunoassay.

Reported in this work is the development and application of a high sensitivity mass spectrometric immunoassay for the quantitative analysis of C-reactive protein from human plasma. Multiplexed affinity retrieval devices and methodology were developed to simultaneously target retinol binding protein, C-reactive protein, serum amyloid P component, as well as an added exogenous internal reference standard (staphylococcal enterotoxin B) for subsequent MALDI-TOF MS analysis. This approach allows for semiquantitative analysis of both retinol binding protein and serum amyloid P component while performing absolute quantitative measurements of C-reactive protein. The ability to qualitatively differentiate between all three human proteins and their associated variants is also maintained. Standard curve, QC, and human plasma samples were analyzed in a high throughput manner, which performed with a CV < 15%. The resultant human plasma sample C-reactive protein quantitative measurements were then compared to those achieved with a high sensitivity latex immunoturbidimetric assay.

C-Reactive Protein↗

PubMed2006

Surface plasmon resonance mass spectrometry for protein analysis.

The combination of surface plasmon pesonance (SPR) and mass spectrometry (MS) provides a unique methodology for studying proteins and their interactions. SPR is utilized to assess protein quantitative variations and the kinetic aspects of protein interactions, whereas MS complements the analysis by providing an exclusive look at the structural features of the interacting proteins via measurement of their mass. Thus, intrinsic protein structural modifications that go unregistered via the SPR detection can readily be assessed from the MS data. The purpose of this chapter is dissemination of the procedures and protocols for successful SPR-MS analysis. The individual steps of the complete SPRMS process are illustrated via analysis of cardiac troponin I (cTnI).

Biosensing Techniques↗

PubMed2006

High-throughput affinity mass spectrometry.

Affinity mass spectrometry (AMS) is a proteomics approach for selectively isolating target protein(s) from complex biological fluids for mass spectrometric analysis. The resulting high-content mass spectrometry (MS) data show the unique MS protein signatures (wild-type, posttranslationally modified, as well as genetically modified forms of the protein target) that are present within a biological sample. Information regarding such protein diversity is normally lost in classical proteomic or immunoassay analyses. This chapter presents a step-by-step description of high-throughput AMS in the population proteomic screening of the human plasma protein cystatin C.

Amino Acid Sequence↗

PubMed2006

Mass spectrometry-based immunoassays for the next phase of clinical applications.

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.

Animals↗

PubMed2005

Investigating diversity in human plasma proteins.

Plasma proteins represent an important part of the human proteome. Although recent proteomics research efforts focus largely on determining the overall number of proteins circulating in plasma, it is equally important to delineate protein variations among individuals, because they can signal the onset of diseases and be used as biological markers in diagnostics. To date, there has been no systematic proteomics effort to characterize the breadth of structural modifications in individual proteins in the general population. In this work, we have undertaken a population proteomics study to define gene- and protein-level diversity that is encountered in the general population. Twenty-five plasma proteins from a cohort of 96 healthy individuals were investigated through affinity-based mass spectrometric assays. A total of 76 structural forms/variants were observed for the 25 proteins within the samples cohort. Posttranslational modifications were detected in 18 proteins, and point mutations were observed in 4 proteins. The frequency of occurrence of these variations was wide-ranged, with some modifications being observed in only one sample, and others detected in all 96 samples. Even though a relatively small cohort of individuals was investigated, the results from this study illustrate the extent of protein diversity in the human population and can be of immediate aid in clinical proteomics/biomarker studies by laying a basal-level statistical foundation from which protein diversity relating to disease can be evaluated.

Adolescent↗

PubMed2005

High-throughput MS-based protein phenotyping: application to haptoglobin.

A high-throughput affinity capture and reduction approach was developed for phenotype and post-translational modification analysis of a complexed globular protein, haptoglobin (Hp), directly from human plasma. Hp was selectively retrieved utilizing anti-Hp antibodies immobilized onto affinity pipette tips, eluted onto a formatted mass spectrometer target for reduction of Hp alpha-chains (Hpalpha1 and Hpalpha2) and subjected to subsequent MALDI-MS analysis. The affinity capture and reduction approach was originally developed from a pre-extraction reduction methodology that was optimized to an affinity capture post-reduction technique for intact Hp alpha-chain variant analysis, phenotype classification and ensuing post-translational variant detection. Three common Hp phenotypes (1-1, 2-1 and 2-2) were assigned according to detection of Hpalpha1 and/or Hpalpha2 reduced intact chain(s) average mass(es). The affinity capture post-reduction approach was scaled for high-throughput Hp alpha-chain phenotype analysis from a normal plasma cohort. The entire sample cohort was successfully analyzed and phenotyped using the developed approach. Additionally, Hp post-translational variants were detected and assigned via accurate MS analyses. The results of this study suggest use of the methodology in future analyses of other similarly complexed proteins and in normal versus disease cohort population proteomics studies.

Haptoglobins↗

PubMed2005

Population proteomics: addressing protein diversity in humans.

In the past several years, proteomics and its subdiscipline clinical proteomics have been engaged in the discovery of the next generation protein of biomarkers. As the effort and the intensive debate it has sparked continue, it is becoming apparent that a paradigm shift is needed in proteomics in order to truly comprehend the complexity of the human proteome and assess its subtle variations among individuals. This review introduces the concept of population proteomics as a future direction in proteomics research. Population proteomics is the study of protein diversity in human populations. High-throughput, top-down mass spectrometric approaches are employed to investigate, define and understand protein diversity and modulations across and within populations. Population proteomics is a discovery-oriented endeavor with a goal of establishing the incidence of protein structural variations and quantitative regulation of these modifications. Assessing human protein variations among and within populations is viewed as a paramount undertaking that can facilitate clinical proteomics' effort in discovery and validation of protein features that can be used as markers for early diagnosis of disease, monitoring of disease progression and assessment of therapy. This review outlines the growing need for analyzing individuals' proteomes and describes the approaches that are likely to be applied in such a population proteomics endeavor.

Adult↗

PubMed2004

High-throughput comprehensive analysis of human plasma proteins: a step toward population proteomics.

A high-throughput (HT) comprehensive analysis approach was developed for assaying proteins directly from human plasma. Proteins were selectively retrieved, by utilizing antibodies immobilized within affinity pipet tips, and eluted onto enzymatically active mass spectrometer targets for subsequent digestion and structural characterization. Several parameters, including uniform parallel protein elution from 96 affinity pipet tips, proper buffering for on-target digestion, termination of the digestion, and MALDI matrix (re)introduction, were evaluated and optimized. The approach was validated via parallel, high-throughput analysis of transthyretin (TTR) and transferrin (TRFE) from 96 identical plasma samples. The 96 parallel analyses for each protein were completed in less than 90 min, measured from protein extraction to insertion in the mass spectrometer. Virtually identical mass spectra were obtained from the 96 TTR analyses, characterized by the presence of 14 tryptic fragments that allowed TTR sequence mapping with 100% coverage. Database search returned TTR as the best match for all 96 data sets. In regard to the TRFE analyses, database searching using data from the 96 spectra returned TRFE as the best match for all but 1 of the spectra. TRFE was mapped with 47-69% sequence coverage, with gaps in the sequence coverage corresponding to the carbohydrate-containing peptide fragments and large and small trypsin fragments that fell outside the window of mass analysis. Overall, the combined high-throughput affinity capture-protein digestion approach showed high reproducibility and speed and yielded an exceptional level of protein characterization, suggesting its use in future population proteomics endeavors.

Blood Proteins↗

PubMed2004

Proteomic characterization of novel serum amyloid P component variants from human plasma and urine.

Serum amyloid P component (SAP) is a human plasma protein that has been widely studied for its influence on amyloid plaque formation and stabilization. SAP was characterized directly from human plasma and urine samples via novel affinity mass spectrometry-based proteomic technology that is able to readily discriminate between mass-altered protein variants. These analyses were able to identify several variants of SAP that have not been previously reported. These variants include microheterogeneity of the glycan structure, from the loss of one or both terminal sialic acid residues, as well as the loss of the C-terminal valine residue. Moreover, the analysis of urine allowed for the consistent identification of serum amyloid P component as a normal constituent of the urine proteome.

Adult↗

PubMed2003

Detection of novel truncated forms of human serum amyloid A protein in human plasma.

Serum amyloid A protein (SAA) is a human plasma protein that has been recognized as potential biomarker of multiple ailments including myocardial infarction, inflammatory disease and amyloiosis. Presented here is the application of a novel immunoassay technique, termed mass spectrometric immunoassay for the detection and identification of SAA present in human plasma. Results demonstrate the ability to readily detect known SAA isotypes, and to identify novel truncated forms of SAA, in the plasma of healthy individuals and those suffering from acute and chronic inflammation. The approach represents a rapid and sensitive means for the routine structural characterization of known SAA isotypes and the discovery of associated post-translational modifications.

Amino Acid Sequence↗

PubMed2003

Detection of bound and free IGF-1 and IGF-2 in human plasma via biomolecular interaction analysis mass spectrometry.

Insulin like growth factor (IGF)-1 and IGF-2 were assayed from human plasma via biomolecular interaction analysis mass spectrometry, utilizing antibodies as ligands for affinity retrieval. Detection of both targeted and non-targeted IGFs in the mass spectra indicated possible protein complex retrieval by the individual antibodies. A series of control experiments eliminated the possibility of analyte cross-walking between flow cells, significant antibodies cross-reactivity, and direct IGF interactions. To disrupt the putative protein complex and release its constituent proteins, plasma samples were treated with detergents. An SDS-treated plasma yielded IGF signals in a different ratio than the one observed in the mass spectra from the non-treated plasma, suggesting disruption of the protein complex, and its retrieval from non-treated plasma. Novel truncated IGF-2 variant, missing its N-terminal Alanine, was detected in all mass spectra.

Humans↗

PubMed2003

Surface plasmon resonance mass spectrometry: recent progress and outlooks.

The combination of surface plasmon resonance (SPR) and mass spectrometry (MS) has created a unique approach to protein investigations. Surface plasmon resonance is used to quantify interactions between proteins and surface-immobilized ligands, and MS is used to determine the structural features of the bound proteins. Recent progress in SPR-MS includes improved methods and operations, increased limits of detection, multi-protein analysis and protein-complex delineation. With the subsequent design of SPR protein arrays, SPR-MS is expected to enter into the field of high-throughput protein interaction discovery and miniaturized diagnostics.

Mass Spectrometry↗

PubMed2003

Detection of Staphylococcal enterotoxin B via biomolecular interaction analysis mass spectrometry.

Detection of Staphylococcus enterotoxin B (SEB) by biomolecular interaction analysis mass spectrometry (BIA/MS) is presented in this work. The BIA/MS experiments were based on a surface plasmon resonance (SPR) MS immunoassay that detects affinity-captured SEB both via SPR and by means of exact and direct mass measurement by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Experiments were performed with standard samples and food samples to assess the BIA/MS limit of detection for SEB and to set the experimental parameters for proper quantitation. Single and double SPR referencing was performed to accurately estimate the amount of the bound toxin. Reproducible detection of 1 ng of SEB per ml, corresponding to affinity capture and MS analysis of approximately 500 amol of SEB, was readily achieved from both the standard and mushroom samples. A certain amount of SEB degradation was indicated by the signals in the mass spectra. The combination of MS with SPR-based methods of detection creates a unique approach capable of quantifying and qualitatively analyzing protein toxins from pathogenic organisms.

Antigens, Bacterial↗