Abstract：
:High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.
journal_name
J Proteome Resjournal_title
Journal of proteome researchauthors
Whiteaker JR,Zhao L,Frisch C,Ylera F,Harth S,Knappik A,Paulovich AGdoi
10.1021/pr4009404subject
Has Abstractpub_date
2014-04-04 00:00:00pages
2187-96issue
4eissn
1535-3893issn
1535-3907journal_volume
13pub_type
杂志文章abstract：:Protein citrullination (or deimination), an irreversible post-translational modification, has been implicated in several physiological and pathological processes, including gene expression regulation, apoptosis, rheumatoid arthritis, and Alzheimer's disease. Several research studies have been carried out on citrullina...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.9b00118
更新日期：2019-05-03 00:00:00
abstract：:Gastric cancer (GC) is among the most common cancers worldwide. Gastric carcinogenesis is a multistep and multifactorial process beginning with chronic gastritis induced by Helicobacter pylori (H. pylori) infection. This process is often described via a sequence of events known as Correas's cascade, a stepwise progres...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.6b00281
更新日期：2016-08-05 00:00:00
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journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.5b00283
更新日期：2015-08-07 00:00:00
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journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.7b00490
更新日期：2017-10-06 00:00:00
abstract：:Traditional measures of short-term stress response such as fecal glucocorticoid metabolites (FGM) are widely used in controlled settings to quantify the intensity of stimulation to which cattle are exposed. However, FGMs present several methodological and interpretation pitfalls when applied on animals in free-ranging...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.9b00655
更新日期：2020-02-07 00:00:00
abstract：:The effects of increased intake of dietary fiber as either whole grain rye or as resistant starch compared to a typical low dietary fiber Western-style diet on the metabolomics responses were studied in gastrointestinal content and tissue, peripheral plasma, and urine using a multicompartmental nontargeted liquid chro...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.5b00039
更新日期：2015-08-07 00:00:00
abstract：:The SwePep database is designed for endogenous peptides and mass spectrometry. It contains information about the peptides such as mass, pl, precursor protein and potential post-translational modifications. Here, we have improved and extended the SwePep database with tandem mass spectra, by adding a locally curated ver...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr800036d
更新日期：2008-07-01 00:00:00
abstract：:The gut microbiota plays a critical role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Increased fructose consumption and inadequate copper intake are two critical risk factors in the development of NAFLD. To gain insight into the role of gut microbiota, fecal metabolites, obtained from rats exposed...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.5b00596
更新日期：2015-09-04 00:00:00
abstract：:As a result of the complexity and dynamic range of the cellular proteome, including mutual interactions and interactions with other molecules, focused proteomic approaches are important to study subsets of physiologically important proteins. In one such approach, a small molecule or part of a protein is immobilized on...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr060278b
更新日期：2006-09-01 00:00:00
abstract：:A major challenge in developing cell therapies is reliable characterization of the cell product at the molecular level. Fresh autologous and passaged human bone marrow enriched for stem and mesenchymal stromal stem cells have been used to regenerate bone. We report the proteome of an innovative autologous human bone m...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr801064u
更新日期：2009-04-01 00:00:00
abstract：:Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the ...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr400877e
更新日期：2013-12-06 00:00:00
abstract：:Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV nonstructural protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influe...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.5b00396
更新日期：2016-05-06 00:00:00
abstract：:The role played by the degree of folding of protein backbones in explaining the binding energetics of protein-ligand interactions has been studied. We analyzed the protein/peptide interactions in the RNase-S system in which amino acids at two positions of the peptide S have been mutated. The global degree of folding o...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr0503174
更新日期：2006-01-01 00:00:00
abstract：:MS-based de novo peptide sequencing has been improved remarkably with significant development of mass-spectrometry and computational approaches but still lacks quality-control methods. Here we proposed a novel algorithm pSite to evaluate the confidence of each amino acid rather than the full-length peptides obtained b...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.7b00428
更新日期：2018-01-05 00:00:00
abstract：:Recoding refers to the reprogramming of mRNA translation by nonstandard read-out rules. In this study, we used stable isotope labeling with amino acids in cell culture (SILAC) technology to investigate the proteome of host-adapted Salmonella serovars, which are characteristic of accumulation of pseudogenes. Interestin...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr200904c
更新日期：2012-03-02 00:00:00
abstract：:Post-translational nitration of proteins was analyzed by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) on-line interfaced to electrospray ionization mass spectrometry (ESI--MS) or tandem mass spectrometry (ESI--MS/MS). Both methods were compared using a tryptic digest of bovine serum albumi...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr034034s
更新日期：2003-09-01 00:00:00
abstract：:Antibody reagents have been remained as a standard approach to characterize blood group (BG) antigens in clinic. The specificity and cross-reactivity of these BG antibodies are routine detected using the gel microcolumn assay (GMA). However, the GMA is neither specific nor sensitive, thus increasing the risk of improp...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.8b00370
更新日期：2018-09-07 00:00:00
abstract：:Histones regulate a variety of chromatin templated events by their post-translational modifications (PTMs). Although there are extensive reports on the PTMs of canonical histones, the information on the histone variants remains very scanty. Here, we report the identification of different PTMs, such as acetylation, met...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr500597a
更新日期：2014-12-05 00:00:00
abstract：:Histone linker proteins H1 and H5 were purified from chicken erythrocyte cell nuclei under nondenaturing conditions. The purified linker histones were analyzed using in-solution enzymatic digestions followed by nanoflow reverse-phase high-performance liquid chromatography tandem mass spectrometry. We have identified a...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr800260a
更新日期：2008-10-01 00:00:00
abstract：:Acute stress may trigger systemic biochemical and physiological changes in living organisms, leading to a rapid loss of homeostasis, which can be gradually reinstated by self-regulatory mechanisms and/or drug intervention strategy. However, such a sophisticated metabolic regulatory process has so far been poorly under...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr070051w
更新日期：2007-09-01 00:00:00
abstract：:To gain insights into how TGF-beta regulates epithelial-mesenchymal transition (EMT), we assessed the time course of proteins and mRNAs during EMT by multiplex iTRAQ labeling and 2D-LC-MS/MS, and by hybridization, respectively. Temporal iTRAQ analysis identified 66 proteins as differentially expressed during EMT, incl...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr8006478
更新日期：2009-01-01 00:00:00
abstract：:Kalirin-7 (Kal7), a multifunctional Rho GDP/GTP exchange factor (GEF) for Rac1 and RhoG, is embedded in the postsynaptic density at excitatory synapses, where it participates in the formation and maintenance of dendritic spines. Kal7 has been implicated in long-term potentiation, fear memories, and addiction-like beha...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr200088w
更新日期：2011-06-03 00:00:00
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journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr050230i
更新日期：2005-11-01 00:00:00
abstract：:Translationally controlled tumor protein (TCTP) is a highly conserved housekeeping protein present in eukaryotic organisms. It is involved in regulating many fundamental processes and plays a critical role in tumor reversion and tumorigenesis. Increasing evidence suggests that TCTP plays a role in the regulation of ce...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.6b00556
更新日期：2016-10-07 00:00:00
abstract：:MHC molecules present protein-derived peptides to T lymphocytes. By developing TiO2-based microcentrifugation columns, we identified the first phosphorylated MHC class I ligands from tumor tissue (renal cell carcinoma) and, by comparison to healthy renal tissue, found one Brf1-derived ligand as potentially tumor-assoc...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr800937k
更新日期：2009-07-01 00:00:00
abstract：:Phosphorylation is an essential post-translational modification for regulating protein function and cellular signal transduction. Mass spectrometry (MS) combined with isobaric tandem mass tags (TMTs) has become a powerful platform for simultaneous, large-scale phospho-proteome site identification and quantitation. To ...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/acs.jproteome.7b00610
更新日期：2017-11-03 00:00:00
abstract：:Antibodies are a primary research tool for a diverse range of experiments in biology, from development to pathology. Their utility is derived from their ability to specifically identify proteins at a high level of sensitivity. This diversity of experimental requirements stretches the capabilities of these key research...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr100029f
更新日期：2010-07-02 00:00:00
abstract：:While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible "window" to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, ...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr8001639
更新日期：2008-08-01 00:00:00
abstract：:New technologies have advanced the field of proteomics, and a number of companies have developed innovative platforms to drive this research. However, significant challenges are often encountered when trying to integrate complementary technologies from multiple manufacturers. We have developed a software and hardware ...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr050465u
更新日期：2006-09-01 00:00:00
abstract：:Hepatitis C virus (HCV) causes chronic liver disease worldwide. HCV Core protein (Core) forms the viral capsid and is crucial for HCV pathogenesis and HCV-induced hepatocellular carcinoma, through its interaction with the host factor proteasome activator PA28γ. Here, using BD-PowerBlot high-throughput Western array, w...
journal_title：Journal of proteome research
pub_type： 杂志文章
doi：10.1021/pr300121a
更新日期：2012-07-06 00:00:00
