PubMed Health⌕ Search

Biomedical subjects

Urban A Kiernan

Publications and source records attributed to Urban A Kiernan.

At least 19 recordsLinked to original sources
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

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

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↗
PubMed2005

Multiplexed DNA sequencing-by-synthesis.

We report a new DNA sequencing-by-synthesis method in which the sequences of DNA templates, hybridized to a surface-immobilized array of DNA primers, are determined by sensing the number of nucleotides by which the primers in each array spot are extended in sequential DNA polymerase-catalyzed nucleotide incorporation reactions, each with a single fluorescein-labeled deoxyribonucleoside triphosphate (dNTP) species. The fluorescein label is destroyed after each readout by a photostimulated reaction with diphenyliodonium chloride. A DNA polymerase with enhanced ability to incorporate, and to extend beyond, modified nucleotides is used. Self-quenching of adjacent fluorescein labels, which impedes readout of homopolymeric runs, is avoided by diluting the labeled dNTP with unlabeled reagent. Misincorporation effects have been quantified and are small; however, low-level contamination of dNTPs with other nucleotides mimics misincorporation and can produce significant false-positive signals. These impurities are removed by polymerase-catalyzed incorporation into complementary "cleaning duplexes." Here, we demonstrate the accurate sequence readout for a small array of known DNA templates, the ability to quantify homopolymeric runs, and a short sequencing example of sections of the wild-type and mutant BRCA1 gene. For a 20,000-spot array, readout rates in excess of 6000 bases per minute are projected.

Amino Acid Sequence↗
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↗
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↗
PubMed2004

Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus.

Few studies have been conducted to identify the extracellular proteins and enzymes secreted by filamentous fungi, particularly with respect to dispensable metabolic pathways. Proteomic analysis has proven to be the most powerful method for identification of proteins in complex mixtures and is suitable for the study of the alteration of protein expression under different environmental conditions. The filamentous fungus Aspergillus flavus can degrade the flavonoid rutin as the only source of carbon via an extracellular enzyme system. In this study, a proteomic analysis was used to differentiate and identify the extracellular rutin-induced and non-induced proteins secreted by A. flavus. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. While 15 rutin-induced proteins and 7 non-induced proteins were identified, more than 90 protein spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced.

Aspergillus flavus↗
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↗
PubMed2002

Novel mass spectrometric immunoassays for the rapid structural characterization of plasma apolipoproteins.

Novel mass spectrometric immunoassays (MSIAs) for the isolation and structural characterization of plasma apolipoprotein A-I (apoA-I), apoA-II, and apoE have been developed. The assays combine selective isolation of apolipoprotein species via affinity capture with mass-specific detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In application, plasma (from 50 microl of whole blood drawn from individuals, using finger lancet) was addressed with affinity pipette tips derivatized with antibodies toward the specific apolipoprotein. The time required for each assay was approximately 15 min, less if assays on multiple individuals were performed in parallel. In a brief study of five individuals, several recently reported apoA-II variants were identified and observed consistently in all individuals. Additionally, the apoE phenotype of E3/E3 was observed in three of the individuals, and E2/E3 and E3/E4 observed in the remaining two individuals, the latter of whom suffers from Alzheimer's disease. Overall, the MSIA approach offers a rapid, sensitive, and highly accurate means of profiling apolipoproteins from small volumes of plasma.

Adult↗
PubMed2002

Comparative phenotypic analyses of human plasma and urinary retinol binding protein using mass spectrometric immunoassay.

Mass spectrometric immunoassay (MSIA) is a proteomics technology that combines the selectivity of affinity capture with the sensitivity and resolution of mass spectrometric detection. This unique approach allows for intact protein identification therefore is readily capable of discriminating between protein variants, i.e., mutations, posttranslational modifications, and truncations. In this work, MSIA is used in the comparative analyses of retinol binding protein (RBP) from the plasma and urine of a small study population. Detailed RBP profiles were obtained from both biological fluids, resulting in the identification of several catabolic RBP products (present in urine) that have not been previously reported. In addition, comparative analysis of urine samples taken from healthy and renally impaired individuals revealed different breakdown profiles. These results illustrate the use of MSIA for the rapid, sensitive, and accurate profiling of RBP both within and between individuals.

Adult↗
PubMed2002

High-throughput protein characterization using mass spectrometric immunoassay.

A high-throughput mass spectrometric immunoassay system for the analysis of proteins directly from plasma is reported. A 96-well format robotic workstation was used to prepare antibody-derivatized affinity pipette tips for subsequent use in the extraction of specific proteins from plasma and deposition onto 96-well format matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) targets. Samples from multiple individuals were screened with regard to the plasma protein transthyretin (TTR), followed by analysis of the same plasma samples for the transthyretin-associated transport protein, retinol-binding protein (RBP). Analyses were able to detect the presence of posttranslationally modified TTR and RBP, as well as a mutation present in the TTR of one individual. Subsequent analyses of wild-type and mutated TTR using enzymatically active MALDI-TOF MS targets were able to identify the site and nature of the point mutation. The approach represents a rapid (approximately 100 samples/2 h, reagent preparation-to-data) and accurate means of characterizing specific proteins present in large numbers of individuals for proteomic and clinical/diagnostic purposes.

Antibody Affinity↗
PubMedDate not supplied

Comparative urine protein phenotyping using mass spectrometric immunoassay.

Reported here, human urine samples were analyzed for beta-2-microglobulin (beta2m), transthyretin (TTR), cystatin C, urine protein 1 (UP1), retinol binding protein (RBP), albumin, transferrin, and human neutrophil defensin peptides (HNP) using mass spectrometric immunoassay (MSIA). MSIA is a unique analytical technique, which allows for the generation of distinct protein profiles of specific target proteins from each subject, which may be subsequently used in comparative protein expression profiling between all subjects. Comparative profiling allows for the rapid identification of variations within individual protein expression profiles. Although the majority of analyses performed in this study revealed homology between study participants, roughly one-quarter showed variation in the protein profiles. Some of these observed variants included a point mutation in TTR, absence of wild-type RBP, monomeric forms UP1, a novel beta2m glycated end product and altered HNP ratios. MSIA has been previously used in the analysis of blood proteins, but this study shows how MSIA easily transitions to the analysis, of urine samples. This study displays how qualitative urine protein differentiation is readily achievable with MSIA and is useful in identifying proteomic differences between subjects that might be otherwise overlooked with other analytical techniques due to complexity of the resulting data or insufficient sensitivity.

DNA Mutational Analysis↗