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The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi-automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.

The urine proteome (urineome) has become an ideal biological fluid for biomarker study and detection due to its rich protein content as well as its ease and abundance in collection. Protein variation in human plasma has been linked to the presence of disease states in humans; however, it stands to reason that the same is true of the equally complex urineome. In this manuscript we present the combination of two proteomics technologies, mass spectrometric immunoassay and a bioreactive probe, for the detection and characterization of the protein variants of transthyretin (TTR) found in human urine. Coupling these two technologies we could precisely identify protein variants within a population of normal individuals. This is the first report of the detailed characterization of urinary TTR using this combination of proteomic analytical techniques, and demonstrates a novel non-invasive methodology for the routine analysis of this clinically significant urine protein target., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Genetic variations and posttranslational modifications give rise to structural diversity in fully expressed human proteins. Structural modifications can also be induced during the life cycle of a protein and can lead to impaired functioning and pathological conditions. Although a large number of protein modifications have been discovered thus far, their incidence among the general population has not been determined. Here we show that human proteins exhibit a wide range of modifications present at various frequencies in the general population. The screening of 1,000 individuals from four geographical regions in the United States for five plasma proteins revealed the existence of 27 protein modifications. Some variants, such as those resulting from oxidation and single amino acid terminal truncations, were observed in the majority of individuals, whereas point mutations and extensive sequence truncations were detected in only a few individuals. Gender correlations were observed for two protein modifications. The data obtained reveal the extent of structural diversity in the general populace and represent the first such catalogue of structural protein modifications. Systematic studies of this kind will help redefine the normal human proteome and reveal the effects of these modifications in pathological processes.

Affinity mass spectrometry is a proteomics approach for selectively isolating target proteins from complex biological fluids for mass spectrometric analysis. When executed in high throughput mode through affinity pipets, the resulting volumetric mass spectrometry arrays enable rapid protein assaying from hundreds of samples. Furthermore, in combination with postcapture proteolytic degradation, this top-down proteomics approach can reveal structural features (i.e., modifications) in the protein sequences that are result of posttranslational modifications and/or point mutations. Described here in greater detail are the individual steps of the high throughput combination of affinity protein capture in antibody-derivatized affinity pipets, protein elution, and protein processing through enzyme-derivatized mass spectrometry targets.

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.

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.

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.

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.

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.

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.

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.

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.

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.

An alkaline phosphatase-bioreactive probe, in which the enzyme is covalently bound to the mass spectrometry target, has been developed for studies of phosphoproteins. The bioreactive probe was used in combination with affinity capture and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to study hydrolysis of several phosphoproteins found in human saliva. Human salivary proteins were extracted from diluted human saliva with immobilized metal-affinity pipettes, which under defined conditions bound the phosphoproteins of interest preferentially over histatins. Phosphoproteins were eluted directly from the affinity pipettes to the bioreactive probe with diluted ammonium hydroxide, which provided conditions appropriate for hydrolysis by the alkaline phosphatase covalently bound to the probe surface. Results indicate the combination of metal-affinity pipette extraction, alkaline phosphatase-bioreactive probes, and matrix-assisted laser desorption/ionization mass spectrometry is an effective way to find and characterize phosphoproteins, known and unknown, in complex mixtures. Facile hydrolysis of human salivary phosphoproteins by the bioreactive probes was readily observed.

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.

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.

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.

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.

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.

The feasibility of buffer exchange in biosensor chip mass spectrometry, along with the construction of base sensor chips and use of alternative chip chemistries, is demonstrated in this work. Beta-2-microglobulin (beta2m) was used as an analyte and captured in the first flow cell (FC1) on the sensor chip surface by an immobilized anti-beta2m antibody. Low pH buffer was then used to elute the captured analyte from the flow cell and route it to a second flow cell (FC2) downstream that served as a cation exchanger that retains the analyte. Following additional washes in FC1, the analyte present in FC2 was either eluted with a higher pH buffer (to demonstrate the possibility of elution into a downstream trypsin flow cell), or it was subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis to verify its presence in FC2. In a separate experiment, a gold-sputtered glass slide (base chip) was activated through a formation of 11-mercaptoundecanoic acid self-assembled monolayer and via reaction with 1,1"-carbonyldiimidazole. The activated chip was placed manually into the biosensor and two surfaces (flow cells) were derivatized with antibodies to beta2m and cystatin C (cysC). To evaluate the chip performance, diluted human urine aliquot was injected over the flow cells. Following the surface plasmon resonance analysis, the chip was MALDI-TOF MS analyzed, yielding signals from beta2m and cysC from their respective flow cells. Artifacts arising from the surface chemistries were not observed in the analysis.

Studies were conducted to optimize matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI TOF MS) in analyzing the composition of nicotinic acetylcholine receptors (nAChR) from Torpedo californica electric tissue in their membrane-bound, detergent-solubilized, and affinity-purified states. Mass spectra obtained from nAChR-rich membrane fractions gave reasonably good representations of protein compositions indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of those same samples. Efficiency of extraction of nAChR from membranes was not markedly different for most detergents, but quality and signal size of mass spectra were clearly influenced by detergent composition and concentration, protein concentration, and MALDI matrix composition. The best spectra, allowing detection and accurate size determinations for samples containing as little as 10 fmol of pure nAChR, were obtained for samples solubilized in Triton X-100 and assayed by use of a sinapinic acid matrix. Although informative spectra could be obtained for nAChR affinity purified on alpha-cobratoxin (Naja naja siamensis) columns and extracted using sinapinic acid, superior spectra with much higher signal:noise were obtained if extraction media contained Triton X-100 or sodium dodecyl sulfate. nAChR subunit masses determined were similar regardless of the membrane-associated, detergent-solubilized, or affinity-purified state of the preparation. These studies illustrate how masses can be determined for nAChR subunits and for other protein components in Torpedo membrane preparations, such as RAPsyn and Na(+)-K(+)-ATPase alpha and beta subunits. They also provide an underpinning for streamlined analysis of the composition of complex transmembrane proteins using MALDI TOF MS., (©2002 Elsevier Science (USA).)

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.

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 approximately 100 samples/h (biological fluid-->data). The quantitative MSIA of beta2m levels present in human plasma samples is given as illustration.

The use of mass spectrometric immunoassay (MSIA) in analyzing beta-2-microglobulin (beta(2)m) present in human biological fluids (tears, saliva, plasma, and urine) is described. Pipettor tips containing porous affinity frits, derivatized with polyclonal anti-beta(2)m immunoglobulin, were manufactured and used to selectively isolate and concentrate beta(2)m from the biofluids, after which matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to detect beta(2)m unambiguously at its characteristic molecular mass. The affinity tips were found rapid to use, requiring approximately 15 min per analysis, and exhibited low nonspecific binding properties that yielded essentially interference-free analyses. The beta(2)m MSIA was made quantitative by inclusion of an internal standard into the analysis for signal normalization. The resulting assay had a Linear dynamic range (R(2) = 0.983) covering a beta(2)m concentration range of 0.010-1.0 mg/L with a standard error of approximately 5%. In application, urine samples from healthy individuals were screened and compared with sample from an individual suffering from renal infection. Results indicated an approximately 30-fold increase in beta(2)m levels in samples taken from the infected individual. During the screening, MSIA was able to distinguish between wild-type and glycosylated forms of beta(2)m, which made possible the accurate quantification of wild-type beta(2)m without interference from glycosylated versions of the protein. These results demonstrate a new approach to the rapid and accurate detection/quantification of beta(2)m present in biological fluids., (Copyright 2001 Academic Press.)

Rapid advances in genomic sequencing, bioinformatics, and analytical instrumentation have created the field of proteomics, which at present is based largely on two-dimensional electrophoresis (2-DE) separation of complex protein mixtures and identification of individual proteins using mass spectrometry. These analyses provide a wealth of data, which upon further evaluation leads to many questions regarding the structure and function of the proteins. The challenge of answering these questions create a need for high-specificity approaches that may be used in the analysis of biomolecular recognition events and interacting partners, and thereby places great demands on general protein characterization instrumentation and the types of analyses they need to perform. Over the past five years we have been actively involved in interfacing two general, instrumental techniques, surface plasmon resonance-biomolecular interaction analysis (SPR-BIA) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, into a single concerted approach for use in the functional and structural characterization of proteins. Reviewed here is the recent progress made using biomolecular interaction analysis - mass spectrometry (BIA-MS) in the detailed characterization of proteins and protein-protein interactions and the development of biosensor chip mass spectrometry (BCMS) as a new chip-based proteomics approach.

The use of biomolecular interaction analysis mass spectrometry to selectively isolate, detect, and characterize epitope-tagged peptides present in total cell lysates is demonstrated. Epitope-tagged tryptic peptides were captured via affinity interactions with either chelated Ni2+ or monoclonal antibodies and detected using surface plasmon resonance biomolecular interaction analysis (SPR-BIA). After SPR-BIA the tagged peptides were either eluted from the biosensor chips for mass spectrometric analysis or analyzed directly from the biosensor chip using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Protein database searches were performed using the masses of the tagged tryptic peptides, resulting in identification of the protein into which the epitope tag was inserted. Detection limits for both SPR-BIA and MALDI-TOF were at the low-femtomole to subfemtomole level. The approach represents a (multiplexed) high-sensitivity chip-based technique capable of identifying epitope-tagged proteins as they are present in complex mixtures.