Targeted measurements of low abundance proteins in complex mixtures are in high demanded in many areas, not the least in clinical applications measuring biomarkers. We here present the novel platform AFFIRM (AFFInity sRM) that utilizes the power of antibody fragments (scFv) to efficiently enrich for target proteins from a complex background and the exquisite specificity of an SRM-MS based detection. To demonstrate the ability of AFFIRM, three target proteins of interest were measured in a serum background in single- and multiplexed experiments in a concentration range of 5-1000 ng/ml. Linear responses were demonstrated down to low ng/ml concentrations with high reproducibility. The platform allows for high throughput measurements in 96-well format and all steps are amendable to automation and scale-up. We believe the use of recombinant antibody technology in combination with SRM MS analysis provides a powerful way to reach sensitivity, specificity and reproducibility as well as the opportunity to build resources for fast on demand implementation of novel assays.
The ability to design and tailor-make antibodies to meet the biophysical demands required by the vast range of current and future antibody-based applications within biotechnology and biomedicine will be essential. In this proof-of-concept study, we have for the first time tailored human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the dock'n'flash technology. In more detail, we have successfully explored the possibility to expand the genetic code of E. coli and introduced the photoreactive UAA p-benzoyl-L-phenylalanine (pBpa), and showed that the mutated scFv antibody could be expressed in E. coli with retained structural and functional properties, as well as binding affinity. The pBpa group was then used for affinity capture of the mutated antibody by ?-cyclodextrin (?-CD), which provided the hydrogen atoms to be abstracted in the subsequent photocoupling process upon irradiation at 365nm. The results showed that the pBpa mutated antibody could be site-specifically photocoupled to free and surface (array) immobilized ?-CD. Taken together, this paves the way for novel means of tailoring recombinant scFv antibodies for site-specific photochemical-based tagging, functionalization and immobilization in numerous applications.
Miniaturized (Ø 10 ?m), multiplexed (>5-plex), and high-density (>100 000 spots cm(-2)) antibody arrays will play a key role in generating protein expression profiles in health and disease. However, producing such antibody arrays is challenging, and it is the type and range of available spotters which set the stage. This pilot study explored the use of a novel microspotting tool, Bioplume(TM)-consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels-to produce miniaturized, multiplexed, and high-density planar recombinant antibody arrays for protein expression profiling which targets crude, directly labelled serum. The results demonstrated that 16-plex recombinant antibody arrays could be produced-based on miniaturized spot features (78.5 um(2), Ø 10 ?m) at a 7-125-times increased spot density (250 000 spots cm(-2)), interfaced with a fluorescent-based read-out. This prototype platform was found to display adequate reproducibility (spot-to-spot) and an assay sensitivity in the pM range. The feasibility of the array platform for serum protein profiling was outlined.
Antibody-based microarrays are a developing tool for high-throughput proteomics in health and disease. However, in order to enable global proteome profiling, novel miniaturized high-density antibody array formats must be developed.
Early detection of prostate cancer (PC) using prostate-specific antigen (PSA) in blood reduces PC-death among unscreened men. However, due to modest specificity of PSA at commonly used cut-offs, there are urgent needs for additional biomarkers contributing enhanced risk classification among men with modestly elevated PSA.
In the quest to decipher disease-associated biomarkers, miniaturized and multiplexed antibody arrays may play a central role in generating protein expression profiles, or protein maps, of crude serum samples. In this conceptual study, we explored a novel, 4-times larger pen design, enabling us to, in a unique manner, simultaneously print 48 different reagents (antibodies) as individual 78.5 ?m(2) (10 ?m in diameter) sized spots at a density of 38,000 spots cm(-2) using dip-pen nanolithography technology. The antibody array set-up was interfaced with a high-resolution fluorescent-based scanner for sensitive sensing. The performance and applicability of this novel 48-plex recombinant antibody array platform design was demonstrated in a first clinical application targeting SLE nephritis, a severe chronic autoimmune connective tissue disorder, as the model disease. To this end, crude, directly biotinylated serum samples were targeted. The results showed that the miniaturized and multiplexed array platform displayed adequate performance, and that SLE-associated serum biomarker panels reflecting the disease process could be deciphered, outlining the use of miniaturized antibody arrays for disease proteomics and biomarker discovery.
Affinity proteomics, represented by antibody arrays, is a multiplex technology for high-throughput protein expression profiling of crude proteomes in a highly specific, sensitive, and miniaturized manner. The antibodies are individually deposited in an ordered pattern, an array, onto a solid support. Next, the sample is added, and any specifically bound proteins are detected and quantified using mainly fluorescence as the mode of detection. The binding pattern is then converted into a relative protein expression map, or protein atlas, delineating the composition of the sample at the molecular level. The technology provides unique opportunities for various applications, such as protein expression profiling, biomarker discovery, disease diagnostics, prognostics, evidence-based therapy selection, and disease monitoring. Here, we describe the generation and use of planar antibody arrays for serum protein profiling.
Systemic lupus erythematosus (SLE) is a severe autoimmune connective tissue disease. Our current knowledge about the serum proteome, or serum biomarker panels, reflecting disease and disease status is still very limited. Affinity proteomics, represented by recombinant antibody arrays, is a novel, multiplex technology for high-throughput protein expression profiling of crude serum proteomes in a highly specific, sensitive, and miniaturized manner. The antibodies are deposited one by one in an ordered pattern, an array, onto a solid support. Next, the sample is added, and any specifically bound proteins are detected and quantified. The binding pattern is then converted into a relative protein expression map, or protein map, deciphering the composition of the sample at the molecular level. The methodology provides unique opportunities for delineating serum biomarkers reflecting SLE, thus paving the way for improved diagnosis, classification, and prognosis.
B-cell lymphoma (BCL) heterogeneity represents a key issue, often making the classification and clinical management of these patients challenging. In this pilot study, we outlined the first resolved view of BCL disease heterogeneity on the protein level by deciphering disease-associated plasma biomarkers, specific for chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma, using recombinant antibody microarrays targeting mainly immunoregulatory proteins. The results showed the BCLs to be heterogeneous, and revealed potential novel subgroups of each BCL. In the case of diffuse large B-cell lymphoma, we also indicated a link between the novel subgroups and survival.
Proteomics, the large-scale analysis of proteins, is a rapidly evolving field with an increasing number of key clinical applications, such as diagnosis, prognosis, and classification. In order to generate complete protein expression profiles, or protein atlases, any crude sample format must be addressable in a rapid, multiplex, and sensitive manner. A common and clinically central sample format, formalin-fixed, paraffin-embedded (FFPE) tissue material, holds great potential as a source for disease-associated biomarker signatures. However, despite major efforts, extraction and subsequent profiling of proteins from FFPE tissue has proven to be challenging. In this proof-of-concept study, we have demonstrated for the first time that proteins could be extracted, labeled, and subsequently profiled in a multiplex, sensitive, and reproducible manner using recombinant scFv antibody microarrays. Thus, we have added FFPE samples to the list of sample formats available for high-throughput analysis by affinity proteomics, paving the way for the next generation of biomarker-driven discovery projects.
Evaluation of: Akada J, Kamei S, Ito A et al. A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients. Proteome Sci. 11(1), 33 (2013). Unlocking the proteome and delivering biomarkers to the clinic will be critical for early and improved diagnosis and prognosis. Conventional protein microarrays have evolved as a promising proteomic technology with great potential for protein expression profiling in health and disease. In this study, Akada et al. explore a new type of protein chip, interfaced with a dual-color fluorescence-based read-out, for screening of autoantibodies in serum. Uniquely, the recombinant antigens were microarray adapted by molecular design to contain a five-cysteine tag for immobilization and green fluorescent protein for detection (color 1). The engineered antigens were immobilized on in-house-designed maleimide-incorporated diamond-like carbon substrates and subsequently heat treated in a solution of denaturing and reducing agents before any specifically bound serum autoantibodies were detected (color 2). The authors used a 4-plex array targeting hepatocellular carcinoma-related autoantibodies in the sera of hepatitis C virus-positive patients as model system to demonstrate proof-of-concept.
Tumor progression and prognosis in breast cancer patients are difficult to assess using current clinical and laboratory parameters, where a pathological grading is indicative of tumor aggressiveness. This grading is based on assessments of nuclear grade, tubule formation, and mitotic rate. We report here the first protein signatures associated with histological grades of breast cancer, determined using a novel affinity proteomics approach. We profiled 52 breast cancer tissue samples by combining nine antibodies and label-free LC-MS/MS, which generated detailed quantified proteomic maps representing 1,388 proteins. The results showed that we could define in-depth molecular portraits of histologically graded breast cancer tumors. Consequently, a 49-plex candidate tissue protein signature was defined that discriminated between histological grades 1, 2, and 3 of breast cancer tumors with high accuracy. Highly biologically relevant proteins were identified, and the differentially expressed proteins indicated further support for the current hypothesis regarding remodeling of the tumor microenvironment during tumor progression. The protein signature was corroborated using meta-analysis of transcriptional profiling data from an independent patient cohort. In addition, the potential for using the markers to estimate the likelihood of long-term metastasis-free survival was also indicated. Taken together, these molecular portraits could pave the way for improved classification and prognostication of breast cancer.
The immobilization of functional biomolecules to surfaces is a critical process for the development of biosensors for disease diagnostics. In this work we report the patterned attachment of single chain fragment variable (scFv) antibodies to the surface of metal oxides by the photodeprotection of self-assembled monolayers, using near-UV light. The photodeprotection step alters the functionality at the surface; revealing amino groups that are utilized to bind biomolecules in the exposed regions of the substrate only. The patterned antibodies are used for the detection of specific disease biomarker proteins in buffer and in complex samples such as human serum.
Affinity proteomics, mainly represented by antibody microarrays, has in recent years been established as a powerful tool for high-throughput (disease) proteomics. The technology can be used to generate detailed protein expression profiles, or protein maps, of focused set of proteins in crude proteomes and potentially even high-resolution portraits of entire proteomes. The technology provides unique opportunities, for example biomarker discovery, disease diagnostics, patient stratification and monitoring of disease, and taking the next steps toward personalized medicine. However, the process of designing high-performing, high-density antibody micro- and nanoarrays has proven to be challenging, requiring truly cross-disciplinary efforts to be adopted. In this mini-review, we address one of these key technological issues, namely, the choice of probe format, and focus on the use of recombinant antibodies vs. polyclonal and monoclonal antibodies for the generation of antibody arrays.
Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.
The risk of distant recurrence in breast cancer patients is difficult to assess with current clinical and histopathological parameters, and no validated serum biomarkers currently exist. Using a recently developed recombinant antibody microarray platform containing 135 antibodies against 65 mainly immunoregulatory proteins, we screened 240 sera from 64 patients with primary breast cancer. This unique longitudinal sample material was collected from each patient between 0 and 36 mo after the primary operation. The velocity for each serum protein was determined by comparing the samples collected at the primary operation and then 3-6 mo later. A 21-protein signature was identified, using leave-one-out cross-validation together with a backward elimination strategy in a training cohort. This signature was tested and evaluated subsequently in an independent test cohort (prevalidation). The risk of developing distant recurrence after primary operation could be assessed for each patient, using her molecular portraits. The results from this prevalidation study showed that patients could be classified into high- versus low-risk groups for developing metastatic breast cancer with a receiver operating characteristic area under the curve of 0.85. This risk assessment was not dependent on the type of adjuvant therapy received by the patients. Even more importantly, we demonstrated that this protein signature provided an added value compared with conventional clinical parameters. Consequently, we present here a candidate serum biomarker signature able to classify patients with primary breast cancer according to their risk of developing distant recurrence, with an accuracy outperforming current procedures.
Antibody-based microarrays are a rapidly evolving affinity-proteomic methodology that recently has shown great promise in clinical applications. The resolution of these proteomic analyses is, however, directly related to the number of data-points, i.e. antibodies, included on the array. Currently, this is a key bottleneck because of limited availability of numerous highly characterized antibodies. Here, we present a conceptually new method, denoted global proteome survey, opening up the possibility to probe any proteome in a species-independent manner while still using a limited set of antibodies. We use context-independent-motif-specific antibodies directed against short amino acid motifs, where each motif is present in up to a few hundred different proteins. First, the digested proteome is exposed to these antibodies, whereby motif-containing peptides are enriched, which then are detected and identified by mass spectrometry. In this study, we profiled extracts from human colon tissue, yeast cells lysate, and mouse liver tissue to demonstrate proof-of-concept.
Systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are two severe autoimmune connective tissue diseases. The fundamental knowledge about their etiology is limited and the conditions display complex pathogenesis, multifaceted presentations, and unpredictable courses. Despite significant efforts, the lack of fully validated biomarkers enabling diagnosis, classification, and monitoring of disease activity represents significant unmet clinical needs. In this discovery study, we have for the first time used recombinant antibody microarrays for miniaturized, multiplexed serum protein profiling of SLE and SSc, targeting mainly immunoregulatory proteins. The data showed that several candidate SLE-associated multiplexed serum biomarker signatures were delineated, reflecting disease (diagnosis), disease severity (phenotypic subsets), and disease activity. Selected differentially expressed markers were validated using orthogonal assays and a second, independent patient cohort. Further, biomarker signatures differentiating SLE versus SSc were demonstrated, and the observed differences increased with severity of SLE. In contrast, the data showed that the serum profiles of SSc versus healthy controls were more similar. Hence, we have shown that affinity proteomics could be used to de-convolute crude, nonfractionated serum proteomes, extracting molecular portraits of SLE and SSc, further enhancing our fundamental understanding of these complex autoimmune conditions.
Generating global protein expression profiles, including also membrane proteins, will be crucial for our understanding of biological processes in health and disease. In this study, we have expanded our antibody microarray technology platform and designed the first human recombinant antibody microarray for membrane proteins targeting crude cell lysates and tissue extracts. We have optimized all key technological parameters and successfully developed a setup for extracting, labeling and analyzing non-fractionated membrane proteomes under non-denaturing conditions. Finally, the platform was also extended and shown to be compatible with simultaneous profiling of both membrane proteins and water-soluble proteins.
In the past decade, many initiatives were taken for the development of antibodies for proteome-wide studies, as well as characterisation and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to antibody generation by immunisation because it is an unlimited resource of affinity reagents without batch-to-batch variation and is also amendable for high throughput in contrast to conventional hybridoma technology. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens for full-length proteins in phage selections for the retrieval of target-specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.
A method called DocknFlash was developed to offer site-specific capture and direct UVA-induced photocoupling of recombinant proteins. The method involves the tagging of recombinant proteins with photoreactive p-benzoyl-L-phenylalanine (pBpa) by genetic engineering. The photoreactive pBpa tag is used for affinity capture of the recombinant protein by beta-cyclodextrin (beta-CD), which provides hydrogen atoms to be abstracted in the photocoupling process. To exemplify the method, a recombinant, folded, and active N27pBpa mutant of cutinase from Fusarium solani pisi was produced in E. coli. Insertion of pBpa was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. A molecular dynamic simulation, with water as solvent, showed high solvent accessibility of the pBpa benzophenone group in N27pBpa-cutinase mutant. The formation of an inclusion complex between the benzophenone group of N27pBpa-cutinase and beta-CD was shown, and an apparent K(d) of 1.65 mM was determined using (1)H NMR. Photocoupling of beta-CD to N27pBpa-cutinase in a 1:1 ratio, upon UVA irradiation at 360 +/- 20 nm, was shown by MALDI-TOF mass spectroscopy. UVA photoimmobilization of N27pBpa-cutinase on quartz slides coated with beta-CD was achieved from liquid or dry films by total internal reflection fluorescence (TIRF). The DocknFlash method offers a solution for direct photocoupling and patterning of recombinant proteins onto surfaces with site-specific attachment.
Glioblastoma multiforme (GBM) is a frequent and aggressive type of primary brain tumor with a heterogeneous origin. GBM is highly therapy resistant and carries a dismal prognosis for the patient. The purpose of this discovery study was to define candidate plasma biomarker signatures for improved classification and novel means for selecting patients for refined individualized therapy.
preeclampsia (PE) is a severe, multi-system pregnancy disorder of yet unknown cause, missing means of treatment, and our fundamental understanding of the disease is still impaired. The purpose of this discovery study was to define candidate placenta tissue protein biomarker signatures to further decipher the molecular features of PE.
Antibody array-based technology is a powerful emerging tool in proteomics, but to enable global proteome analysis, antibody array layouts with even higher density has to be developed. To this end, we have further developed the first generation of a nanoarray platform, based on attoliter-sized vials, attovials, which we have characterized and used for the detection of complement factor C1q in human serum samples. Finally, we demonstrated proof-of-concept for individual functionalization of the attovials with a recombinant antibody.
Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.
Dendritic cells (DCs) are central in allergy as regulators of the Th1/Th2 balance. We have recently demonstrated a unique transcriptional profile of DCs in patients with ongoing allergy compared with healthy subjects and shown that crosstalk between DCs and memory T cells affects the transcriptional profile of T cells. However, the transcriptional profile of DCs educated by T cells in allergy is unknown.
Antibody-based microarrays is a rapidly evolving technology that has gone from the first proof-of-concept studies to more demanding proteome profiling applications, during the last years. Miniaturized microarrays can be printed with large number of antibodies harbouring predetermined specificities, capable of targeting high- as well as low-abundant analytes in complex, nonfractionated proteomes. Consequently, the resolution of such proteome profiling efforts correlate directly to the number of antibodies included, which today is a key limiting factor. To overcome this bottleneck and to be able to perform in-depth global proteome surveys, we propose to interface affinity proteomics with MS-based read-out, as outlined in this technical perspective. Briefly, we have defined a range of peptide motifs, each motif being present in 5-100 different proteins. In this manner, 100 antibodies, binding 100 different motifs commonly distributed among different proteins, would potentially target a protein cluster of 10(4) individual molecules, i.e. around 50% of the nonredundant human proteome. Notably, these motif-specific antibodies would be directly applicable to any proteome in a specie independent manner and not biased towards abundant proteins or certain protein classes. The biological sample is digested, exposed to these immobilized antibodies, whereby motif-containing peptides are specifically captured, enriched and subsequently detected and identified using MS.
Antibody-based microarrays are a new powerful proteomic technology that can be used to generate rapid and detailed expression profiles of defined sets of protein analytes in complex samples as well as high-resolution portraits of entire proteomes. Miniaturized micro- and nanoarrays can be printed with numerous antibodies carrying the desired specificities. Multiplexed and ultra-sensitive assays, specifically targeting several analytes in a single experiment, can be performed, while consuming only minute amounts of the sample. The array images generated can then be converted into protein expression profiles, or maps, revealing the detailed composition of the sample. This promising proteomic research tool will thus provide unique opportunities for e.g. disease proteomics, biomarker discovery, disease diagnostics, and patient stratification. This review describes the antibody-based microarray technology and applications thereof.
Antibody-based microarray is a novel proteomic technology setting a new standard for molecular profiling of non-fractionated complex proteomes. The first generation of antibody microarrays has already demonstrated its potential for generating detailed protein expression profiles, or protein atlases, of human body fluids in health and disease, paving the way for new discoveries within the field of disease proteomics. The process of designing highly miniaturized, high-density and high-performing antibody microarray set-ups have, however, proven to be challenging. In this mini-review we discuss key technological issues that must be addressed in a cross-disciplinary manner before true global proteome analysis can be performed using antibody microarrays.
Protein-peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein-peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody-peptide interaction characteristics, by combining large-scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide-binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low-affinity antibody-peptide interactions. The molecular mechanism for the degenerate peptide-binding specificity appeared to be executed through the use of 2-3 semi-conserved anchor residues in the C-terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex-peptide complexes. In the long-term, this knowledge will be instrumental for advancing our fundamental understanding of protein-peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide-binding proteins in general, in various biotechnical and medical applications.
Pancreatitis is an inflammatory state of the pancreas, for which high-performing serological biomarkers are lacking. The aim of the present study was to evaluate the use of affinity proteomics for identifying potential markers of disease and stratifying pancreatitis subtypes.
Urinary proteomics has become a key discipline within clinical proteomics for noninvasive diagnosis and monitoring of disease, and biomarker discovery. In order to decipher complex proteomes, high demands will, however, be placed upon the methodology applied. The purpose of this study was to develop a recombinant antibody microarray platform for urinary proteomics.
Pancreatic cancer is an aggressive disease with poor prognosis, due, in part, to the lack of disease-specific biomarkers that could afford early and accurate diagnosis. With a recombinant antibody microarray platform, targeting mainly immunoregulatory proteins, we screened sera from 148 patients with pancreatic cancer, chronic pancreatitis, autoimmune pancreatitis (AIP), and healthy controls (N). Serum biomarker signatures were derived from training cohorts and the predictive power was evaluated using independent test cohorts. The results identified serum portraits distinguishing pancreatic cancer from N [receiver operating characteristics area under the curve (AUC) of 0.95], chronic pancreatitis (0.86), and AIP (0.99). Importantly, a 25-serum biomarker signature discriminating pancreatic cancer from the combined group of N, chronic pancreatitis, and AIP was determined. This signature exhibited a high diagnostic potential (AUC of 0.88). In summary, we present the first prevalidated, multiplexed serum biomarker signature for diagnosis of pancreatic cancer that may improve diagnosis and prevention in premalignant diseases and in screening of high-risk individuals.
The development of high-performance technology platforms for generating detailed protein expression profiles, or protein atlases, is essential. Recently, we presented a novel platform that we termed global proteome survey, where we combined the best features of affinity proteomics and mass spectrometry, to probe any proteome in a species independent manner while still using a limited set of antibodies. We used so called context-independent-motif-specific antibodies, directed against short amino acid motifs. This enabled enrichment of motif-containing peptides from a digested proteome, which then were detected and identified by mass spectrometry. In this study, we have demonstrated the quantitative capability, reproducibility, sensitivity, and coverage of the global proteome survey technology by targeting stable isotope labeling with amino acids in cell culture-labeled yeast cultures cultivated in glucose or ethanol. The data showed that a wide range of motif-containing peptides (proteins) could be detected, identified, and quantified in a highly reproducible manner. On average, each of six different motif-specific antibodies was found to target about 75 different motif-containing proteins. Furthermore, peptides originating from proteins spanning in abundance from over a million down to less than 50 copies per cell, could be targeted. It is worth noting that a significant set of peptides previously not reported in the PeptideAtlas database was among the profiled targets. The quantitative data corroborated well with the corresponding data generated after conventional strong cation exchange fractionation of the same samples. Finally, several differentially expressed proteins, with both known and unknown functions, many relevant for the central carbon metabolism, could be detected in the glucose- versus ethanol-cultivated yeast. Taken together, the study demonstrated the potential of our immunoaffinity-based mass spectrometry platform for reproducible quantitative proteomics targeting classes of motif-containing peptides.
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