Selected Reaction Monitoring Method Research Articles

Rapid and Quantitative Analysis of Aflatoxin M1 From Milk Using Atmospheric Pressure-Matrix Assisted Laser Desorption/Ionization (AP-MALDI)-Triple Quadrupole Selected Reaction Monitoring.

Aflatoxin M1 (AFM1) is a carcinogenic hydroxylated metabolite commonly found in milk. It is relatively stable toward decontamination procedures posing a major health risk, and it requires an international regulatory mandate of detection at trace levels. To develop a high-throughput, reliable, and compliant method for the identification of AFM1 in milk samples using atmospheric pressure-matrix assisted laser desorption/ionization (AP-MALDI) selected reaction monitoring (SRM) quantitation. The milk sample was diluted in water and cleaned with immunoaffinity chromatography (IAC), followed by analysis using AP-MALDI hyphenated with a triple quadrupole mass spectrometer for SRM. A fast and reliable AP-MALDI SRM quantitative method was developed for the determination of AFM1 with analysis time of 1 min per sample. The diagnostic product ions of AFM1 at 273.1 u and 229.2 u were monitored during the SRM. The calibration curves yielded excellent linearity (R2 = 0.99) with good recoveries for quality control samples (97-106%). The ion ratios of the qualifier to quantifier displayed excellent RSD (1-7.8%) for n = 3. The developed method provided rapid quantification for AFM1. The fast AP-MALDI SRM method can allow analysis of AFM1 in a large number of milk samples. Given the time required for analysis, cost-effectiveness, and superior analytical performance, this method can be adopted in commercial food testing laboratories. Aflatoxins (AF) are a major health risk. Speedy analysis of large sample sizes from food is a risk mitigation strategy but remains an unmet need. Quantitative, chromatography-free, and internal standard-free AP-MALDI SRM based analysis of AF is a high-throughput and cost-efficient alternative. Satisfactory performance was achieved for quantitative AP-MALDI SRM analysis of AFM1 in milk subsequent to a simple sample clean-up step.

Generation and Release of Neurogranin, Vimentin, and MBP Proteolytic Peptides, Following Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major neurological disorder without FDA-approved therapies. In this study, we have examined the concept that TBI might trigger global brain proteolysis in the acute post-injury phase. Thus, we conducted a systemic proteolytic peptidomics analysis using acute cerebrospinal fluid (CSF) samples from TBI patients and normal control samples. We employed ultrafiltration-based low molecular weight (LMW; < 10kDa) peptide enrichment, coupled with nano-reversed-phase liquid chromatography/tandem mass spectrometry analysis, followed with orthogonal quantitative immunoblotting-based protein degradation analysis. We indeed identified novel patterns of injury-dependent proteolytic peptides derived from neuronal components (pre- and post-synaptic terminal, dendrites, axons), extracellular matrix, oligodendrocytes, microglial cells, and astrocytes. Among these, post-synaptic protein neurogranin was identified for the first time converted to neurogranin peptides including neurogranin peptide (aa 16-64) that is phosphorylated at Ser-36/48 (P-NG-fragment) in acute human TBI CSF samples vs. normal control with a receiver operating characteristic area under the curve of 0.957. We also identified detailed processing of astroglia protein (vimentin) and oligodendrocyte protein (MBP and Golli-MBP) to protein breakdown products (BDPs) and/or LMW proteolytic peptides after TBI. In addition, using MS/MS selected reaction monitoring method, two C-terminally released MBP peptides TQDENPVVHFF and TQDENPVVHF were found to be elevated in acute and subacute TBI CSF samples as compared to their normal control counterparts. These findings imply that future therapeutic strategies might be placed on the suppression of brain proteolysis as a target. The endogenous proteolytic peptides discovered in human TBI biofluid could represent useful diagnostic and monitoring tools for TBI.

Development and Interlaboratory Evaluation of an LC-MS/MS Method for the Quantification of Lysozyme in Wine Across Independent Instrument Platforms.

Various processing aids and fining agents are used in winemaking to help improve sensory characteristics. Some of these materials may contain or be derived from allergenic foods, such as eggs. In order to ensure food safety and that products meet regulatory compliance, it is essential to have robust and effective analytical methods to verify the removal of allergenic proteins following their use. Current methods include ELISA and MS methods, which can target either whole foods or individual proteins, and provide either quantitative data or qualitative confirmation of proteins. MS methods offer the potential to test for multiple proteins within a single assay to improve cost and efficiency, whereas ELISA methods typically analyze for a single protein per assay. This study focuses on the development of a LC-tandem MS (MS/MS) quantitative method for lysozyme in white wine and compares performance across two laboratories utilizing two different instrument platforms. Lysozyme target peptides were selected by conducting bottom-up discovery proteomics. Candidate targets were evaluated using parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) LC-MS/MS, depending on the instrument in each laboratory. Quantification of lysozyme was conducted using internal, stable isotope-labeled synthetic peptide standards. Three of eight candidate target peptides showed performance suitable for the final quantitative method. White wine spiked with 0.1 and 0.5 ppm lysozyme demonstrated quantitative recovery of 70-120%. While the PRM method delivered better repeatability, the SRM method gave higher quantitative recovery values. A targeted LC-MS/MS method for quantification of lysozyme in white wine has been developed and deployed on two different MS instrument platforms in two laboratories. Both SRM and PRM targeted LC-MS/MS methodologies can be used for quantification of lysozyme in white wine. This study is among the first to evaluate an MS method for food allergen quantification in multiple laboratories.

Selected Reaction Monitoring Method for The Quantification of Escherichia coli Ribosomal Proteins

【Research objective】 Due to the constraints regarding cell growth, modification of ribosomal genes in vivo is challenging. The reconstruction of E. coli ribosomes in vitro will be an important step to engineer ribosomes. We are trying to reconstruct the ribosome biogenesis process in E. coli lysates by expressing all of the ribosomal components so that the transcription, translation, and assembly of ribosomal building blocks proceed cooperatively. For the improvement and the validation of this strategy, analytical tools for ribosomal proteins (r-proteins) are necessary. From this point of view, we tried to develop a highly-specific method for the quantification of all of the ribosomal proteins (r-proteins) by using selected reaction monitoring (SRM). The SRM methods will enable highly specific quantification of r-proteins even if their concentrations are very low in samples. 【Methods】 In this study, we analyzed r-proteins derived from purified E. coli ribosome, E. coli lysate, and r-protein-overexpressed E. coli lysate using nanoLC–MS/MS (LCMS-8060) and developed optimized SRM methods for each r-protein. 【Results & Discussion】 Using the SRM methods, we successfully quantified 41 of 54 nascent r-proteins synthesized in E. coli lysate. To improve production efficiencies of r-proteins, we optimized translation enhancer region of the plasmids encoding r-proteins. In conclusion, we successfully developed a highly-specific and comprehensive analytical method for r-proteins, which will promote the study of ribosome reconstruction as well as the understanding of ribosome biogenesis.

Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins.

Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.

Discovery based high resolution MS/MS analysis for selection of allergen markers in chocolate and broth powder matrices

Peptide marker identification is an important step in development of a mass spectrometry method for multiple allergen detection, since specificity, robustness and sensitivity of the overall analytical method will depend on the reliability of the proteotypic peptides. As part of the development of a multi-analyte reference method, discovery analysis of two incurred food matrices has been undertaken to select the most reliable peptide markers. Six allergenic ingredients (milk, egg, peanut, soybean, hazelnut, and almond) were incurred into either chocolate or broth powder matrix. Different conditions of protein extraction and purification were tested and the tryptic peptide pools were analysed by untargeted high resolution tandem mass spectrometry and the resulting fragmentation spectra were processed via a commercial software for sequence identification. The analysis performed on incurred foods provides both a prototype effective and straightforward sample preparation protocol and delivers reliable peptides to be included in a standardized selected reaction monitoring method.

A high throughput approach for determination of dermorphin in human urine using liquid chromatography-mass spectrometry for doping control purposes.

Dermorphin is a peptide with analgesic actions similar to morphine, but with greater effect and less potential to cause tolerance. The use of dermorphin has been documented in race horses, and its use in humans has already been reported. Considering the potential advantages from the use of dermorphin over morphine, a method to monitor it, and its main metabolite dermorphin (1-4) in humans becomes necessary for doping control. Here, we present two orthogonal methods for this purpose: a high-throughput liquid chromatography coupled to high-resolution mass spectrometry (HRMS) as an initial testing procedure and liquid chromatography-tandem mass spectrometry (MS/MS) in the selected reaction monitoring (SRM) acquisition mode for a confirmation procedure. For urine samples, pretreatment through a mixed-mode weak cation-exchange solid-phase extraction emerged as an effective approach to extract peptides from the biological sample. For the HRMS analysis, a full-MS scan acquisition mode was selected to detect the exact masses of dermorphin and dermorphin (1-4) at m/z 803.37226 and 457.20816, respectively. The SRM method used in the MS/MS confirmation protocol presented high specificity and sensitivity. The selected product ions for dermorphin were 602.2, 202.1 and 574.3 and for dermorphin (1-4) were 207.1, 223.1, and 235.1. Both methods were evaluated for specificity, repeatability, carryover, matrix effects, and recovery. No carryover and matrix effects were detected. The limit of detection for initial testing procedure and the limit of identification for confirmation procedure was 2.5 ng/ml. Also, specificity and robustness were acceptable for the application. Together, the developed methods proved to be efficient for the analysis of dermorphin and metabolite for human doping control purpose.

Lipid alterations in the skeletal muscle tissues of mice after weight regain by feeding a high-fat diet using nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry

This study investigated lipid alterations in muscle tissues [gastrocnemius (Gas) and soleus (Sol)] of mice under different diet programs (weight gain, weight maintenance, weight regain, and controls) by nanoflow ultrahigh pressure liquid chromatography-electrospray ionization-tandem mass spectrometry. Since overloaded lipids in the skeletal muscle tissues by excessive fat accumulation are related to insulin resistance leading to type II diabetes mellitus, analysis of lipid alteration in muscle tissues with respect to high-fat diet (HFD) is important to understand obesity related diseases. A total of 345 individual lipid species were identified with their molecular structures, and 184 lipids were quantified by selected reaction monitoring method. Most triacylglycerol (TG) and phosphatidylethanolamine (PE) species displayed a significant (>2-fold, p < 0.01) increase in both the Gas and Sol and to a larger degree in the Gas. However, lipid classes involved in insulin resistance and anti-inflammatory response, including lysophosphatidylcholine (18:0), diacylglycerol (16:0_18:1, 16:0_18:2, and 18:1_18:1), ceramide (d18:1/24:0 and d18:1/24:1), and phosphatidylinositol (18:0/20:4), showed a significant accumulation in the Sol exclusively after HFD treatment. In addition, the lipid profiles were not significantly altered in mice that were fed HFD only for the last 4 weeks (weight gain group), suggesting that consuming HFD in the younger age period can be more effective in the Gas. This study reveals that lipid classes related to insulin resistance accumulated more in the Sol than in the Gas following HFD treatment and the weight regain program perturbed lipid profiles of the Sol to a greater extent than that by the other diet programs, confirming that the Sol tissue is more influenced by HFD than Gas.

Permanent-Charge Generation Derivatization for the Analysis of Thiols by “Soft” Ionization Mass Spectrometry Methods

A new method is proposed for the permanent-charge generation derivatization of thiols of different structures by a reaction with haloalkanes and dibromides. It was shown that 1,4-dibromobutane and 1,2-xylylene dibromide are the most effective alkylating agents, providing the formation of a stable five-membered ring with a positively charged sulfur atom. Such cations are readily detected by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry. Sulfonium cations were further studied by tandem mass spectrometry using collision-induced dissociation (CID). Typical and specific CID directions for cyclic cations formed from thiols of different structures are revealed. They can be used to identify and quantify thiol analytes by the selected reaction monitoring method.

Development and validation of a UHPLC-MS/MS method for the identification of irinotecan photodegradation products in water samples

Irinotecan (CPT-11) is a water-soluble anticancer drug widely used to treat several types of cancer.Even if the metabolites of CPT-11 are well-known and investigated, only limited information is available in the literature about the formation of photo-degradation products that can naturally originate from sunlight irradiation when the drug is released in aqueous systems.CTP-11 solutions at 10.0 mg L−1 were irradiated by simulated sunlight. The intensity of the drug decreased by 90% after 7.5 days of irradiation and no significant reduction of absorbance values was observed after 13 days.A sensitive UHPLC-MS/MS method was developed employing a hybrid triple quadrupole/linear ion trap mass spectrometer, that is able to work in data-dependent acquisition mode and to obtain information about the compounds formed during the photoirradiation. Moreover, a selected reaction monitoring method was built using the MS/MS fragmentation pattern of the compounds previously investigated. The method was validated considering LOD, LOQ, linearity, precision, selectivity, recovery and matrix effect. LOD and LOQ values were 0.02 and 0.05 ng mL−1, respectively, whereas MDL and MQL values in real water samples (river water, groundwater, well water, and wastewater) were lower than 0.05 and 0.2 ng mL−1, respectively.Eight photodegradation products were identified, among which five for the first time. Based on the MS and MS/MS fragmentation, the chemical structures of the degradation products were proposed. Hydrolysis experiments were carried out on the same solutions preserved in the dark, but no formation of other species was highlighted.The method was applied to several real samples: CPT-11 was detected and quantified only in a hospital effluent sample at the concentration of 0.41 ± 0.2 ng mL−1 together with the occurrence of PDP3.The outcomes of this study may be useful for updating the pollutant screening in water samples.

Evaluation of meter-long monolithic columns for selected reaction monitoring mass spectrometry

Proteome is extremely complex as many proteins with a large dynamic range are involved. Nano-liquid chromatography/mass spectrometry-based proteomics has made it possible to separate and identify thousands of proteins in one shot. Although the number of identified proteins in proteomics has significantly improved, it is necessary to increase detection sensitivity to clearly identify low-abundant proteins. In this study, we developed meter-long monolithic columns with a small inner diameter and applied them to selected reaction monitoring-based proteomics for improving proteomic detection sensitivity. Bovine serum albumin tryptic digests were analyzed with optimized selected reaction monitoring methods, and separation efficiency and detection sensitivity in each monolithic column were evaluated. As a result, peak capacity increased by about 1.8-fold and peak area of peptide levels increased by about 2.3-fold. Although flow rate was reduced during analysis with columns of a smaller inner diameter, the peak area reproducibility was maintained. These data displayed the value of meter-long monolithic columns with small inner diameter for selected reaction monitoring-based proteomics.

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.

Analysis of lipoprotein-specific lipids in patients with acute coronary syndrome by asymmetrical flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry

A comprehensive lipid analysis was performed at the plasma lipoprotein level in patients with acute coronary syndrome (ACS) and stable coronary artery disease (CAD). Because the lipids in lipoproteins are related to the pathology of the cardiovascular system, lipoprotein-specific lipid analysis can be useful for understanding the mechanism of lipid-associated cardiovascular diseases. Lipoproteins were size-sorted into high density lipoproteins (HDL) and low density lipoproteins (LDL) using asymmetrical flow field-flow fractionation, then lipids of each lipoprotein were analysed using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry. A total of 365 lipids were structurally identified and quantified by selected reaction monitoring method. Two high abundance lysophosphatidylcholines (16:0 and 18:0) were significantly increased only in the HDL of the ACS group (vs. the stable CAD group). Phosphatidylethanolamines (38:5 and 40:5) significantly increased in ACS by >2-fold in both lipoproteins. (18:0, 22:6)-diacylglycerol increased in ACS by 3.5-fold only in LDL; however, most high abundance triacylglycerols decreased 2-fold in both lipoproteins. The present study revealed the usefulness of lipoprotein-specific analysis of lipids in distinguishing ACS from stable CAD, and the selected lipids analysed in this study may be useful in the development of lipid markers for the early detection of ACS.

Picky: a simple online PRM and SRM method designer for targeted proteomics.

Targeted proteomic approaches like selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) are increasingly popular because they enable sensitive and rapid analysis of a preselected set of proteins. However, developing targeted assays is tedious and requires the selection, synthesis and mass spectrometric analysis of candidate peptides before the actual samples can be analyzed. The SRMatlas and ProteomeTools projects recently published fragmentation spectra of synthetic peptides covering the entire human proteome. These datasets provide very valuable resources. However, extracting the relevant data for selected proteins of interest is not straightforward. For example, developing scheduled acquisition methods (i.e. analyzing specific peptides in defined elution time windows) is complicated and requires adjustments to specific chromatographic conditions employed. Here, we present Picky: a fast and easy to use online tool to design scheduled PRM/SRM assays (https://picky.mdc-berlin.de). Picky (i) automatically generates optimized and scheduled SRM/PRM assays for proteins of interest and (ii) provides means to validate the data via known fragmentation spectra of corresponding synthetic peptides.

Algorithm of targeted (SRM) methods development in mass-spectrometric studies

Determination of protein concentration in biological samples is an important task for biological research, as well as for medicine and routine clinical biochemistry. The introduction of stable isotope-labeled peptide standards (SIS) made it possible to determine accurately absolute protein concentrations in proteomic studies. The correct choice of SIS and the systematic way to develop a s method for selected reaction monitoring (SRM) are very important steps that are crucial for further identification and measurements of protein concentration. In this paper, we summarize our experience of selecting SIS for measuring the protein concentration by SRM. The results are presented in the form of an algorithm that describes the main stages of the SIS selection and the main points in the development of SRM methods for the targeted protein detection and determination of protein concentrations in biological samples.

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean).

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

Quantification of Von Willebrand Factor Cleavage by adamts-13 in Patients Supported by Left Ventricular Assist Devices.

Patients supported by left ventricular assist devices (LVADs) often present with the loss of large von Willebrand factor (VWF) multimers. This VWF deficiency is believed to contribute to the bleeding diathesis of patients on LVAD support and is caused by excessive VWF cleavage by the metalloprotease ADAMTS-13 under high shear stress. However, only a small percentage of patients who have suffered the loss of large VWF multimers bleed. The actual rates of VWF cleavage in these patients have not been reported, primarily because of the lack of reliable detection methods. We have developed and validated a selected reaction monitoring (SRM) mass spectrometry method to quantify VWF cleavage as the ratio of the ADAMTS-13-cleaved peptide MVTGNPASDEIK to the ILAGPAGDSNVVK peptide. The rate of VWF cleavage was found to be 1.26% ± 0.36% in normal plasma. It varied significantly in patient samples, ranging from 0.23% to 2.5% of total VWF antigen, even though all patients had the loss of large VWF multimers. Von Willebrand factor cleavage was greater in post-LVAD samples from patients in whom bleeding had developed, but was mostly reduced in patients in whom thrombosis had developed. This SRM method is reliable to quantify the rate of VWF cleavage in patients on LVAD support.

Ionization and collision induced dissociation of steroid bisglucuronides.

Studies on steroid metabolism are of utmost importance to improve the detection capabilities of anabolic androgenic steroids (AASs) misuse in sports drug testing. In humans, glucuronoconjugates are the most abundant phase II metabolites of AAS. Bisglucuronidation is a reaction where two separated functional groups on the same molecule are conjugated with glucuronic acid. These metabolites have not been studied in depth for steroids and could be interesting markers for doping control. The aim of the present work was to study the ionization and collision-induced dissociation of steroid bisglucuronides to be able to develop mass spectrometric analytical strategies for their detection in urine samples after AAS administration. Because steroid bisglucuronides are not commercially available, 19 of them were qualitatively synthesized to study their mass spectrometric behavior. Bisglucuronides ionized as [M+NH4 ]+ in positive mode, and as [M-H]- and [M-2H]2- in negative mode. The most specific product ions of steroid bisglucuronides in positive mode resulted from the neutral losses of 387 and 405Da (corresponding to [M+NH4 -NH3 -2gluc-H2 O]+ and [M+NH4 -NH3 -2gluc-2H2 O]+ , respectively, being "gluc" a dehydrated glucuronide moiety), and in negative mode, the fragmentation of [M-2H]2- showed ion losses of m/z 175 and 75 (gluc- and HOCH2 CO2- , respectively). On the basis of the common behavior, a selected reaction monitoring method was developed to detect bisglucuronide metabolites in urine samples. As a proof of concept, urines obtained after administration of norandrostenediol were studied, and a bisglucuronide metabolite was detected in those urines. The results demonstrate the usefulness of the analytical strategy to detect bisglucuronide metabolites in urine samples, and the formation of these metabolites after administration of AAS.

Quantitative LC/ESI-SRM/MS of antibody biopharmaceuticals: use of a homologous antibody as an internal standard and three-step method development.

Monoclonal antibody-based therapeutic agents (antibody drugs) have attracted considerable attention as a new type of drug. Concomitantly, the use of quantitative approaches for characterizing antibody drugs, such as liquid chromatography (LC)-mass spectrometry (MS), has increased. Generally, selective quantification of antibody drugs is done using unique peptides from variable regions (V H and V L) as surrogate peptides. Further, numerous internal standards (ISs) such as stable isotope-labeled (SIL)-intact proteins and SIL-surrogate peptides are used. However, developing LC-MS methodology for characterizing antibody drugs is time-consuming and costly. Therefore, LC-MS is difficult to apply for this purpose, particularly during the drug discovery stage when numerous candidates must be evaluated. Here, we demonstrate an efficient approach to developing a quantitative LC/electrospray ionization (ESI)-selected reaction monitoring (SRM)/MS method for characterizing antibody drugs. The approach consists of the following features: (i) standard peptides or SIL-IS are not required; (ii) a peptide from the homologous monoclonal antibody serves as an IS; (iii) method development is monitored using a spiked plasma sample and one quantitative MS analysis; and (iv) three predicted SRM assays are performed to optimize quantitative SRM conditions such as transition, collision energy, and declustering potential values. Using this strategy, we developed quantitative SRM methods for infliximab, alemtuzumab, and bevacizumab with sufficient precision (<20%)/accuracy (<±20%) for use in the drug discovery stage. We have also demonstrated that choosing a higher homologous peptide pair (from analyte mAb/IS mAb) is necessary to obtain the sufficient precision and accuracy. Graphical abstract ᅟ.

Strategy for the Quantitation of a Protein Conjugate via Hybrid Immunocapture-Liquid Chromatography with Sequential HRMS and SRM-Based LC-MS/MS Analyses.

With the development of modern instrumentation and technologies, mass spectrometry based assays have played an important role in protein bioanalysis. We have developed a novel strategy by combining the "bottom-up" and "top-down" approaches using both high-resolution (HRMS) and selected reaction monitoring (SRM) based mass spectrometric detection to quantify a positron emission tomography (PET) detection tracer for an oncology marker. Monkey plasma samples were processed by immunocapture purification, followed by liquid chromatography (LC) with HRMS full scan analysis. Summed multiple charge states and multiple isotopes per charge state of the analyte were used during quantitation for optimized sensitivity. After the HRMS analysis, the remaining samples were digested by trypsin, followed by SRM detection. The HRMS approach provided the solution to a unique problem related to stability of the protein conjugate by quantifying the intact protein. The SRM method only measured a signature peptide generated from enzymatic digestion, but had a lower quantitation limit to meet the sensitivity requirement to assess the pharmacokinetics in a toxicology study. Both methods demonstrated good sensitivity, accuracy, precision and robustness, and the results revealed that there was no significant difference between the data sets obtained from both methods, indicating no in vivo or ex vivo degradation occurred in the incurred samples after dosing. This workflow not only provided the quantitative results for pharmacokinetic evaluation, but also revealed valuable in vivo stability information on the intact protein level.