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Protein microarrays are the format of choice for high-throughput, high-content protein interaction analysis. In most of the array formats, reporter molecules are used in multistep detection of the protein interactions. Among the few existing label-free detection approaches, surface plasmon resonance (SPR) and mass spectrometry (MS) stand out as most promising for utilization in protein microarrays, albeit both have been used only sporadically for high-content protein arrays. Shown here for the first time is the combination of SPR and MS detection on a single high-content protein microarray. Antibodies to five human plasma proteins were arrayed in a 10 x 10 spot arrangement on a chemically activated gold-coated glass chip. Binding of proteins to their corresponding antibodies was monitored via SPR imaging across the entire surface of the chip. Following protein affinity retrieval, the chip was overlaid with MALDI matrix and MS analyzed, producing protein-specific mass spectra from distinct spots on the array. The SPR-MS dual detection is well suited for high-content protein microarrays and comprehensive protein analysis-from quantitative assessment of the protein concentration to detection of structural protein variants arising from genetic variations and postexpression processing.

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 [micro]m down to 200 [micro]m. As a demonstration, a three-component antibody array was employed to detect two clinically important protein biomarkers, [[beta].sub.2]-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.

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 resisfin related disease cohorts.

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.

A high-throughput mass spectrometric immunoassay (MSIA) system for the analysis of proteins directly from biological fluids is reported. A 96-well-format robotic workstation equipped with antibody-derivatized affinity pipet tips was used for the parallel extraction of specific proteins from samples and subsequent deposition onto 96-well arrayed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) targets. Interferences from nonspecifically bound proteins were minimized through choice of appropriate affinity pipet tip derivatization chemistries. Sample preparation for MALDI-TOFMS was enhanced through the use of hydrophobic/hydrophilic contrasting targets, which also presented functionalities found to promote matrix/analyte crystal growth. Automated mass spectrometry was used in the unattended acquisition of data, resulting in an analysis rate of ~100 samples/h (biological fluid [right arrow] data). The quantitative MSIA of [Beta.sub.2]m levels present in human plasma samples is given as illustration.

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 Fr-IR reflection absorption spectroscopy. The CDI chemistry was then employed to create an antibody microarray with array element sizes varying from 750 μm down to 200 pm. As a demonstration, a three-component antibody array was employed to detect two clinically important protein biomarkers, β2-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. [ABSTRACT FROM AUTHOR]

Describes the root techniques of biomolecular interaction analysis mass spectrometry (BIA/MS). Surface plasmon resonance biomolecular interaction analysis; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Optimization of the interface of BIA/MS; Multi-analyte assays; Ligand fishing/mass profiling.

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.

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.

Reported in this work are the development of mass spectrometric immunoassay (MSIA) devices and methods for the qualitative analysis of IGF-1 and -2, and the rigorous quantification of IGF-1 from human plasma. A method involving addition of SDS in moderate concentration to unfractionated plasma for disrupting IGF/IGFBP complexes was initially developed. The method is suitable for the direct extraction of the IGFs and subsequent mass spectrometric analysis. Rat plasma, containing IGF-1 that is mass shifted from human IGF-1, was used as an internal reference standard (IRS) for the quantification of IGF-1 directly from human plasma. A standard curve with linear dynamic range of at least 2 orders of magnitude was constructed from serially diluted IGF-1 standards containing equal amounts of rat plasma. Using the standard curve, IGF-1 levels in plasma samples from eight individuals were determined. The limit of detection for the IGF-1 MSIA was also evaluated and established to be approximately 15 pM. The assay is rapid and can be performed in parallel via high-throughput robotics processing. Furthermore, the mass spectrometry aspect of the developed IGF-1 immunoassay offers a new dimension in the ongoing study of IGF-1 and related diseases.

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.