Quantitative Analysis Of Peptides Research Articles

Efficient Quality Control of Peptide Pools by UHPLC and Simultaneous UV and HRMS Detection

Peptide pools consist of short amino acid sequences and have proven to be versatile tools in various research areas in immunology and clinical applications. They are commercially available in many different compositions and variants. However, unlike other reagents that consist of only one or a few compounds, peptide pools are highly complex products which makes their quality control a major challenge. Quantitative peptide analysis usually requires sophisticated methods, in most cases isotope-labeled standards and reference materials. Usually, this would be prohibitively laborious and expensive. Therefore, an approach is needed to provide a practical and feasible method for quality control of peptide pools. With insufficient quality control, the use of such products could lead to incorrect experimental results, worsening the well-known reproducibility crisis in the biomedical sciences. Here we propose the use of ultra-high performance liquid chromatography (UHPLC) with two detectors, a standard UV detector at 214 nm for quantitative analysis and a high-resolution mass spectrometer (HRMS) for identity confirmation. To be cost-efficient and fast, quantification and identification are performed in one chromatographic run. An optimized protocol is shown, and different peak integration methods are compared and discussed. This work was performed using a peptide pool known as CEF advanced, which consists of 32 peptides derived from cytomegalovirus (CMV), Epstein–Barr virus (EBV) and influenza virus, ranging from 8 to 12 amino acids in length.

Albumen and Yolk Plasma Peptidomics for the Identification and Quantitation of Bioactive Molecules and the Quality Control of Hen Egg Products.

Hen eggs and the correspondingfood products are essential components of human diet. In addition to supplying basic nutrients, they contain functional peptides that are released in vivo within the intact raw material following physiological proteolytic events affecting specific proteins or derive from technological processing of albumen and yolk fractions as a result of the dedicated use of proteases from plant and microbial sources. Besides their potential importance for functional applications, peptides released under physiological conditions in intact egg can be used as markers of product storage and deterioration. Therefore, characterization and quantitation of peptides in egg and egg-derived products can be used to implement evaluation of potential bioactivities as well as to assess food product qualitative characteristics. Here, we provide dedicated information on extraction, identification, and quantitative analysis of peptides from albumen and yolk plasma; nano-liquid chromatography-mass spectrometry combined with bioinformatic analysis of resulting raw data by different software tools allowed to assign molecules based on database searching and to evaluate their relative quantity in different samples.

Application of peptide biomarkers in life analysis based on liquid chromatography-mass spectrometry technology.

Biomedicine is developing rapidly in the 21st century. Among them, the qualitative and quantitative analysis of peptide biomarkers is of considerable importance for the diagnosis and therapy of diseases and the quality evaluation of drugs and food. The identification and quantitative analysis of peptides have been going on for decades. Traditionally, immunoassays or biological assays are generally used to quantify peptides in biological matrices. However, the selectivity and sensitivity of these methods cannot meet the requirements of the application. The separation and analysis technique of liquid chromatography-mass spectrometry (LC-MS) supplies a reliable alternative. In contrast to immunoassays, LC-MS methods are capable of providing the analytical prowess necessary to satisfy the demands of peptide biomarker research in the life sciences arena. This review article provides a historical account of the in-roads made by LC-MS technology for the detection of peptide biomarkers in the past 10 years, with the focus on the qualification/quantification developments and their applications.

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies.

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

Quantitative analysis of peptides and proteins as biomarkers in biological samples by peptide adsorption-controlled (PAC)-LC/MS

In the pharmaceutical industry, various compounds such as antibodies, peptides, oligonucleotides, etc. are being evaluated and developed as new modality drugs as well as small molecule compounds. For the evaluation of these new modalities, the development of analytical methods is essential, but often difficult because the analytical techniques related to these modalities are not as mature as to small molecule compounds. Liquid chromatography/mass spectrometry (LC/MS) is a highly selective and sensitive method with a wide dynamic range, and has become an indispensable instrument for analysis of small molecule compounds. Recently, LC/MS has attracted attention as an alternative to ligand-biding assays for the quantification of peptides and proteins. So far, we have found that the adsorption capacity of a peptide changes abruptly and reversibly around a specific organic solvent content (critical point) in the solution, and based on this property we developed a peptide adsorption-controlled liquid chromatography (PAC-LC) for quantitative peptide analysis. By using PAC-LC, it has become possible to quantify low concentration proteins and peptides in biological samples. In this symposium, I would like to introduce examples of analysis for biomarker proteins and peptides in biological samples using PAC-LC.

Quantitative Mass Spectrometry-Based Proteomics: An Overview.

In recent decades, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quantification of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review actual strategies and problems of the methods for the quantitative analysis of peptides, proteins, and finally proteomes by mass spectrometry. The common themes, the differences, and the potential pitfalls of the main approaches are presented in order to provide a survey of the emerging field of quantitative, mass spectrometry-based proteomics.

Simultaneously quantitative analysis of peptides and chemical components in Cervus and Cucumis polypeptide injection (Songmeile®) using reversed phase liquid chromatography-hydrophilic interaction liquid chromatography–tandem mass spectrometry

Co-occurrences of peptides and chemical components are usually observed in complicated matrices. Notably, those traditional Chinese medicine prescriptions (TCMPs) contain both plant and animal ingredients. It is still challenging to simultaneously monitor peptides and chemical components attributing to their different liquid chromatographic (LC) and mass spectrometric (MS) behaviors. Herein, efforts were made to pursue an eligible approach enabling simultaneous determination of peptides and chemical components in a TCMP namely Cervus and Cucumis polypeptide injection (CCPI, Songmeile®) that is prepared from the acid hydrolytic peptide-enriched extract of Sika deer (Cervus nippon Temminck) bone and the aqueous extract of muskmelon (Cucumis melo L.) seeds. Reversed phase liquid chromatography and hydrophilic interaction liquid chromatography were serially connected (RPLC-HILIC) to achieve comprehensive retention and separation. Sensitive detection was accomplished with selected reaction monitoring (SRM) mode, and multiply charged and singly charged ion transitions were defined for peptides and chemical components, respectively. Inter-batch variations of CCPI were evaluated in an authentic compound-independent manner. In particular, online energy-resolved MS was proposed to gain optimal parameters for five targeted peptides after that CCPI peptidome was profiled using nanoLC-LTQ Orbitrap Velos Pro MS. A so-called universal metabolome standard (UMS) sample was built for calibration curve construction and subsequently applied to acquire the quasi-contents of all 31 analytes, including five peptides and 26 chemical components, in ten batches of CCPI (CCPI1–CCPI10). The quantitative dataset revealed mild fluctuation for the quasi-content profiles of analytes-of-interest within different batches. More importantly, RPLC-HILIC–SRM is a promising method to fully address the demands of simultaneous measurement of peptides and chemical components in complicated matrices, and it might be a robust analytical tool for in-depth quality evaluation of CCPI as well as other TCMPs.

Self-assembly of super-hydrophobic PMMA microspheres on silicon wafer as MALDI-MS chip for rapid quantification of peptides

Rapid quantification of peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a challenge. Herein, we prepared a super-hydrophobic poly(methyl methacrylate) (PMMA) structured chip for quantifying peptides without internal standard by improving the detection reproducibility and sensitivity. The chip was formed by PMMA microspheres through a self-assembly method. As a good adsorbent of peptides, PMMA microspheres make peptides distributed uniformly on the chip, resulting in the elimination of the coffee ring effect and the homogeneous co-crystallization of peptides/matrix. The relative standard deviations (∼ 10 %) and coefficients of determination (∼ 0.99) are satisfactory when detecting 3 standard peptides, which show that the chip is applicable for rapid quantification of peptides. The limit of detection is improved by 1000-fold on this chip compared with conventional steel target because of the enrichment of the super-hydrophobicity. It can be concluded that the detection reproducibility and sensitivity are significantly improved by the super-hydrophobic structured-PMMA chip. The applicability of the chip for practical samples is demonstrated by quantifying bacitracin A in milk extract and bradykinin1-7 in serum with good linearities. The structured-PMMA chips exhibit competitive advantages for quantitative analysis of peptides, which have a great potential for proteomic research.

High‐Throughput Bioassays using “Dip‐and‐Go” Multiplexed Electrospray Mass Spectrometry

AbstractA multiplexed system based on inductive nanoelectrospray mass spectrometry (nESI‐MS) has been developed for high‐throughput screening (HTS) bioassays. This system combines inductive nESI and field amplification micro‐electrophoresis to achieve a “dip‐and‐go” sample loading and purification strategy that enables nESI‐MS based HTS assays in 96‐well microtiter plates. The combination of inductive nESI and micro‐electrophoresis makes it possible to perform efficient in situ separations and clean‐up of biological samples. The sensitivity of the system is such that quantitative analysis of peptides from 1–10 000 nm can be performed in a biological matrix. A prototype of the automation system has been developed to handle 12 samples (one row of a microtiter plate) at a time. The sample loading and electrophoretic clean‐up of biosamples can be done in parallel within 20 s followed by MS analysis at a rate of 1.3 to 3.5 s per sample. The system was used successfully for the quantitative analysis of BACE1‐catalyzed peptide hydrolysis, a prototypical HTS assay of relevance to drug discovery. IC50 values for this system were in agreement with LC‐MS but recorded in times more than an order of magnitude shorter.

High-Throughput Bioassays using "Dip-and-Go" Multiplexed Electrospray Mass Spectrometry.

A multiplexed system based on inductive nanoelectrospray mass spectrometry (nESI-MS) has been developed for high-throughput screening (HTS) bioassays. This system combines inductive nESI and field amplification micro-electrophoresis to achieve a "dip-and-go" sample loading and purification strategy that enables nESI-MS based HTS assays in 96-well microtiter plates. The combination of inductive nESI and micro-electrophoresis makes it possible to perform efficient in situ separations and clean-up of biological samples. The sensitivity of the system is such that quantitative analysis of peptides from 1-10 000 nm can be performed in a biological matrix. A prototype of the automation system has been developed to handle 12 samples (one row of a microtiter plate) at a time. The sample loading and electrophoretic clean-up of biosamples can be done in parallel within 20 s followed by MS analysis at a rate of 1.3 to 3.5 s per sample. The system was used successfully for the quantitative analysis of BACE1-catalyzed peptide hydrolysis, a prototypical HTS assay of relevance to drug discovery. IC50 values for this system were in agreement with LC-MS but recorded in times more than an order of magnitude shorter.

The impact of environmental factors on the production of peptides in mammalian decomposition fluid in relation to the estimation of post-mortem interval: A summer/winter comparison in Western Australia

We report the peptide content of decomposition fluid produced under field-based conditions and in the absence of a soil matrix. Sixteen domestic pig (Sus scrofa domesticus) cadavers were used to model human decomposition in trials conducted in the summer and winter months in Western Australia. Physical characteristics were recorded and the peptide components of decomposition fluid were analysed using high performance liquid chromatography-time of flight mass spectrometry. A range of peptides was consistently detected in both summer and winter. Thirty seven peptides were common to both trials; 22 originating from haemoglobin subunit beta, 1 from haemoglobin subunit alpha, 4 from beta-enolase, and 2 from creatine kinase. In agreement with our previous findings, 13 peptides occurred consistently, regardless of trial conditions. Degradation patterns for haemoglobin subunits alpha and beta in summer and winter were similar when expressed in ADD and when adjusted for differences in temperature. The consistent identification of several protein-specific peptides generated during decomposition trials conducted under different temperature and rainfall regimes suggests that quantitative peptide analysis may be useful in estimating time since death.

Understanding glycation kinetics of individual peptides in protein hydrolysates

Protein hydrolysates contain peptides with different lengths, type (α/ε-) and number of amino groups; these properties might influence the peptide glycation kinetics during the Maillard reaction. To identify the effects of peptide properties and hydrolysate composition on glycation kinetics, the glycation kinetics of individual peptides in hydrolysates was followed using quantitative peptide analysis. α-Lactalbumin was hydrolysed and glycated with d-glucose (0–8 h, 50 °C, dry heating with 65% humidity). The hydrolysates (degree of hydrolysis 2, 4, 6, and 8%) contained 25 unique peptides, ranging from 2 to 123 AAs with 0–12 lysine(s). The glycation rate constant (kg) and the maximum average degree of glycation (DG_Pav,max) of peptides were independent of the hydrolysate composition. The maximum DG of α-NH2 and ε-NH2 groups was 12.8% and 60.0%, respectively. With this information, the DG_Pav,max of individual peptides [9–59% for peptides with 0–2 lysine(s)] could be predicted.

Sensoproteomics: A New Approach for the Identification of Taste-Active Peptides in Fermented Foods.

Aiming at the identification of the key bitter peptides in fermented foods, a new approach, coined "sensoproteomics", was developed and applied to fresh cheese samples differing in bitter taste intensity. By means of MPLC fractionation of the water-soluble cheese extracts in combination with taste dilution analysis, complex fractions with intense bitter taste were located and then screened by UPLC-MS/MS for the entire repertoire of ∼1600 candidate peptides, extracted from a literature meta-analysis on dairy products, by using a total of 120 selected reaction monitoring methods computed in silico. A total of 340 out of the 1600 peptides were found in the cheese samples, among which 17 peptides were identified as candidate bitter peptides by considering only peptides that were located in the bitter-tasting MPLC fractions (signal-to-noise ratio: ≥10) with a fold-change of ≥3 when comparing the less bitter to the more bitter cheese sample and that were validated by comparison with the synthetic reference peptides. While EIVPNS[phos]VEQK (αs1-CN70-78) and INTIASGEPT (κ-CN122-131) did not exhibit any bitter taste up to 2000 μmol/L, 15 of the 17 target peptides showed bitter taste thresholds ranging from 30 (ARHPHPHLSFM, κ-CN96-106) to 690 μmol/L (IQKEDVPS, αs1-CN81-88). Finally, quantitative peptide analysis followed by calculation of dose-overthreshold factors revealed a primary contribution of MAPKHKEMPFPKYPVEPF (β-CN102-119) and ARHPHPHLSFM (κ-CN96-106) to the perceived bitter taste of the fresh cheese samples. Finally, the evolution of the bitter peptides throughout two different fresh cheese manufacturing processes was quantitatively recorded.

Quantitative Analysis of Peptide-Matrix Interactions in Lyophilized Solids Using Photolytic Labeling.

Peptide-matrix interactions in lyophilized solids were explored using photolytic labeling with reversed phase high performance liquid chromatography (rp-HPLC) and mass spectrometric (MS) analysis. A model peptide (Ac-QELHKLQ-NHCH3) derived from salmon calcitonin was first labeled with a heterobifunctional cross-linker NHS-diazirine (succinimidyl 4,4'-azipentanoate; SDA) at Lys5 in solution, with ∼100% conversion. The SDA labeled peptide was then formulated with the following excipients at a 1:400 molar ratio and lyophilized: sucrose, trehalose, mannitol, histidine, arginine, urea, and NaCl. The lyophilized samples and corresponding solution controls were exposed to UV at 365 nm to induce photolytic labeling, and the products were identified by MS and quantified with rp-HPLC or MS. Peptide-excipient adducts were detected in the lyophilized solids except the NaCl formulation. With the exception of the histidine formulation, peptide-excipient adducts were not detected in solution and the fractional conversion to peptide-water adducts in solution was significantly greater than in lyophilized solids, as expected. In lyophilized solids, the fractional conversion to peptide-water adducts was poorly correlated with bulk moisture content, suggesting that the local water content near the labeled lysine residue differs from the measured bulk average. In lyophilized solids, the fractional conversion to peptide-excipient adducts was assessed using MS extracted ion chromatograms (EIC); subject to the assumption of equal ionization efficiencies, the fractional conversion to excipient adducts varied with excipient type. The results demonstrate that the local environment near the lysine residue of the peptide in the lyophilized solids can be quantitatively probed with a photolytic labeling method.

Prediction of Response to Sorafenib in Hepatocellular Carcinoma: A Putative Marker Panel by Multiple Reaction Monitoring-Mass Spectrometry (MRM-MS).

Sorafenib is the only standard treatment for unresectable hepatocellular carcinoma (HCC), but it provides modest survival benefits over placebo, necessitating predictive biomarkers of the response to sorafenib. Serum samples were obtained from 115 consecutive patients with HCC before sorafenib treatment and analyzed by multiple reaction monitoring-mass spectrometry (MRM-MS) and ELISA to quantify candidate biomarkers. We verified a triple-marker panel to be predictive of the response to sorafenib by MRM-MS, comprising CD5 antigen-like (CD5L), immunoglobulin J (IGJ), and galectin-3-binding protein (LGALS3BP), in HCC patients. This panel was a significant predictor (AUROC > 0.950) of the response to sorafenib treatment, having the best cut-off value (0.4) by multivariate analysis. In the training set, patients who exceeded this cut-off value had significantly better overall survival (median, 21.4 months) than those with lower values (median, 8.6 months; p = 0.001). Further, a value that was lower than this cutoff was an independent predictor of poor overall survival [hazard ratio (HR), 2.728; 95% confidence interval (CI), 1.312-5.672; p = 0.007] and remained an independent predictive factor of rapid progression (HR, 2.631; 95% CI, 1.448-4.780; p = 0.002). When applied to the independent validation set, levels of the cut-off value for triple-marker panel maintained their prognostic value for poor clinical outcomes. On the contrast, the triple-marker panel was not a prognostic factor for patients who were treated with transarterial chemoembolization (TACE). The discriminatory signature of a triple-marker panel provides new insights into targeted proteomic biomarkers for individualized sorafenib therapy.

Synthesis of d-labeled and unlabeled ethyl succinic anhydrides and application to quantitative analysis of peptides by isotope differential mass spectrometry

Ethyl succinic anhydride and its d5-labeled version have been synthesized and applied to quantitative analysis of peptides in combination with MALDI or ESI mass spectrometry. These modifiers react with amino groups in the N-termini and lysine side chains in proteins, and therefore the combination of these modifiers was shown to be a useful tool for quantification of peptides and hence for proteomics research.

Targeted Proteomics Identifies Paraoxonase/Arylesterase 1 (PON1) and Apolipoprotein Cs as Potential Risk Factors for Hypoalphalipoproteinemia in Diabetic Subjects Treated with Fenofibrate and Rosiglitazone

Low levels of high-density lipoprotein cholesterol (HDL-C) and high triglyceride levels contribute to the excess rate of cardiovascular events seen in subjects with type 2 diabetes. Fenofibrate treatment partially reverses dyslipidemia in these subjects. However, a paradoxical marked reduction in HDL-C and HDL's major protein, apolipoprotein A-I, is a complication of fenofibrate in combination with rosiglitazone, an insulin-sensitizing agent. Risk factors for this condition, termed hypoalphalipoproteinemia, have yet to be identified. Using a case-control study design with subjects enrolled in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial, we tested the hypothesis that alterations in HDL's protein cargo predispose diabetic subjects to fenofibrate/rosiglitazone-induced hypoalphalipoproteinemia. HDL was isolated from blood obtained from controls (no decreases or increase in HDL-C while receiving fenofibrate/rosiglitazone therapy) and cases (developed hypoalphalipoproteinemia after fenofibrate/rosiglitazone treatment) participating in the ACCORD study before they began fenofibrate/rosiglitazone treatment. HDL proteins were quantified by targeted parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) with isotope dilution. This approach demonstrated marked increases in the relative concentrations of paraoxonase/arylesterase 1 (PON1), apolipoprotein C-II (APOC2), apolipoprotein C-I, and apolipoprotein H in the HDL of subjects who developed hypoalphalipoproteinemia. The case and control subjects did not differ significantly in baseline HDL-C levels or other traditional lipid risk factors. We used orthogonal biochemical techniques to confirm increased levels of PON1 and APOC2. Our observations suggest that an imbalance in HDL proteins predisposes diabetic subjects to develop hypoalphalipoproteinemia on fenofibrate/rosiglitazone therapy.

Complementary protein and peptide OFFGEL fractionation for high-throughput proteomic analysis.

OFFGEL fractionation of mouse kidney protein lysate and its tryptic peptide digest has been examined in this study for better understanding the differences between protein and peptide fractionation methods and attaining maximum recruitment of this modern methodology for in-depth proteomic analysis. With the same initial protein/peptide load for both fractionation methods, protein OFFGEL fractionation showed a preponderance in terms of protein identification, fractionation efficiency, and focusing resolution, while peptide OFFGEL was better in recovery, number of peptide matches, and protein coverage. This result suggests that the protein fractionation method is more suitable for shotgun analysis while peptide fractionation suits well quantitative peptide analysis [isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT)]. Taken together, utilization of the advantages of both fractionation approaches could be attained by coupling both methods to be applied on complex biological tissue. A typical result is shown in this article by identification of 8262 confident proteins of whole mouse kidney under stringent condition. We therefore consider OFFGEL fractionation as an effective and efficient addition to both label-free and quantitative label proteomics workflow.

Cleavage of host cytokeratin-6 by lysine-specific gingipain induces gingival inflammation in periodontitis patients.

Background/PurposeLysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion.MethodsK6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay.ResultsWe identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359–378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt.ConclusionKgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.

Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach.