Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) ß-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG ß-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10 ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Sample Preparation and Phosphopeptide Enrichment for Plant Phosphoproteomics via Label-Free Mass Spectrometry.

Phosphopeptide enrichment is the main bottleneck of every phosphorylation study. Therefore, in this chapter, a general workflow tries to overbridge the hurdles of plant sample handling from sample collection to protein extraction, protein solubilization, enzymatic digestion, and enrichment step prior to mass spectrometry. The workflow provides information to perform global proteomics as well as phosphoproteomics enabling the researcher to use the protocol in both fields.

Quantification of the lung cancer tumor marker CYFRA 21-1 using protein precipitation, immunoaffinity bottom-up LC-MS/MS.

OBJECTIVES: Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. METHODS: Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. RESULTS: Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0-100 ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. CONCLUSIONS: A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.

Alpha-1-Antitrypsin (A1AT) Proteotyping by LC-MS/MS.

The diagnosis of alpha-1-antitrypsin (A1AT) deficiency is established by quantitation of protein concentration in serum (immunoassay) followed by determination of specific allelic variants by phenotyping (isoelectric focusing (IEF) gel electrophoresis) and/or allele-specific genotyping. Various phenotyping and genotyping methodologies are available, and each has their own advantages and disadvantages. As an alternative, mass spectrometry is emerging as a powerful tool in the identification and quantitation of proteins and peptides. The method described here, referred to as proteotyping, is a proteomic method using trypsin digestion and tandem mass spectrometry that detects the most common deficiency alleles, S and Z, associated with A1AT deficiency.This qualitative mass spectrometry method is based on the principle that the S and Z mutations lead to amino acid changes which result in a change in the mass of the A1AT protein. When the A1AT protein is proteolytically digested, multiple peptides are generated, two of which include the sites of the S and Z mutations, respectively. Peptides generated from wild-type A1AT (M alleles) differ in sequence and mass from peptides generated from the S and Z alleles at these two specific locations. The mass difference allows for differentiation of S and Z peptides, representing the deficiency alleles, from non-S and non-Z peptides, representing the wild-type alleles (M). Interpretation of the peptide patterns in conjunction with A1AT quantitation by immunoassay allows for an accurate assessment for the presence of deficiency alleles in the majority of patients.

Analysis of Neuron-Specific enolase isozymes in human serum using immunoaffinity purification and liquid chromatography-tandem mass spectrometry quantification.

Neuron-specific enolase (NSE) is a promising small-cell lung cancer (SCLC) biomarker composed of αγ and γγ isozyme dimers. As the conventional immunoassays are prone to interferences and cannot differentiate between the isozymes, we developed a multiplex immunoaffinity (IA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of NSEα and NSEγ in human serum. A calibrator was prepared by performing cold denaturation of recombinantly expressed αα and γγ enolase dimers to induce a new dimer equilibrium that was determined to be approximately 1αγ:1γγ:1αα. Selective sample purification was achieved by performing IA extraction using an antibody specific towards NSEγ. The isolated αγ and γγ dimers were denatured and trypsin digested to allow quantification of the selected signature peptides and their corresponding isotopically labelled peptide internal standard. The obtained linear dynamic ranges were determined to be 1.5-56 ng/mL and 0.64-167 ng/mL for NSEα and NSEγ (R2 = 0.88 and 0.97 respectively). Validation of the assay showed acceptable accuracy and precision for NSEα and NSEγ. The method was successfully applied to patient serum in which both isozymes were detected. Compared to the conventional immunoassay, substantially lower total NSE concentrations were measured in IA LC-MS/MS. With this multiplex IA LC-MS/MS assay, the clinical value of quantifying the individual isozymes can be explored. In addition, together with the calibrator described here, it may be applied to standardize NSE immunoassays across different platforms.

Proteomic analysis of Penicillin G acylases and resulting residues in semi-synthetic ß-lactam antibiotics using liquid chromatography - tandem mass spectrometry.

Penicillin G acylase (PGA), as a key enzyme, is increasingly used in the commercial production of semi-synthetic ß-lactam antibiotics (SSBAs). With the substitution of conventional chemical synthesis by emerging bioconversion processes, more and more PGAs fermented from different types of strains such as Escherichia coli (E. coli, ATCC 11105), Achromobacter sp. CCM 4824 and Providencia rettgeri (ATCC 31052) have been used in this kind of enzymatic processes. As an intermediate reaction catalyst, PGA protein and its presence in the final products may cause a potential risk of human allergic reaction and bring challenges for both quality and process controls. To achieve qualitative and quantitative analysis of PGAs and their residues in SSBAs, a tryptic digestion coupled with liquid chromatography - tandem mass spectrometry (LC-MS/MS) method was developed and proposed because of advantages like high selectivity and sensitivity. A suitable filter aided sample preparation (FASP) method was also used to remove matrix interference and to enrich the target PGA retained in the ultrafiltration membrane for an efficient enzymatic hydrolysis and subsequent accurate MS detection. Finally, twelve batches of PGAs from eight companies were identified and categorized into two types of strains (E. coli and Achromobacter sp. CCM 4824) using proteomic analysis. In total nine batches of five types of SSBAs (amoxicillin, cephalexin, cefprozil, cefdinir and cefaclor) from eight manufacturers were selected for investigation. Trace levels of PGA residual proteins ranging from 0.01 to 0.44 ppm were detected in six batches of different SSBAs which were far lower than the safety limit of 35 ppm reported by DSM, a manufacturer with expertise in the production of SSBAs by enzymatic processes. The developed FASP with LC-MS/MS method is superior to traditional protein assays in terms of selectivity, sensitivity and accuracy. Moreover, it could provide in-depth analysis of amino acid sequences and signature peptides contributing to assignment of the strain sources of PGAs. This method could become a promising and powerful tool to monitor enzymatic process robustness and reliability of this kind of SSBAs manufacturing.

Molecular Characterization of Outer Capsid Proteins VP5 and VP7 of Grass Carp Reovirus.

Aquareovirus, which is a member of the Reoviridae family, was isolated from aquatic animals. A close molecular evolutionary relationship between aquareoviruses and mammalian orthoreoviruses was revealed. However, the functions of the aquareovirus genome-encoded proteins are poorly understood. We investigated the molecular characteristics of the outer capsid proteins, namely, VP5 and VP7, of grass carp reovirus (GCRV). The peptides VP5 and VP7 were determined using in-gel tryptic digestion and mass spectrometry. Recovered peptides represented 76% and 66% of the full-length VP5 and VP7 sequences, respectively. Significantly, two-lysine acetylation, as well as two-serine and two-threonine phosphorylation modifications, were first revealed in VP5. We found that the initial amino acid in VP5 was Pro43, suggesting that a lower amount of VP5 remained uncleaved in virions at the autocleavage site (Asn42-Pro43). Further biochemical evidence showed that the cleaved VP5N/VP5C conformation was the major constituent of the particles. Moreover, early cleavage fragments of VP7 and enhanced infectivity were detected after limited tryptic digestion of GCRV, indicating that stepwise VP7 cleavage is essential for VP5 conformational rearrangement. Our results provide insights into the roles of posttranslational modifications in VP5 and its association with VP7 in the viral life cycle.

High-Performance Thin-Layer Chromatography-Immunostaining as a Technique for the Characterization of Whey Protein Enrichment in Edam Cheese.

Whey protein-enriched cheese can be produced by means of a high-temperature treatment of a part of the cheese milk. In this way, the nutritional quality of the resulting cheeses can be increased while resources are conserved. High-performance thin-layer chromatography-immunostaining (HPTLC-IS) using specific ß-lactoglobulin (ß-LG) antibodies was applied to study the implementation and stability of ß-LG in two different sample sets of whey protein-enriched Edam model cheeses, including industrial-scale ones. Two methods were compared for the extraction of the proteins/peptides from the cheese samples. By applying tryptic hydrolysis directly from a suspended cheese sample instead of a supernatant of a centrifuged suspension, a better yield was obtained for the extraction of ß-LG. When applying this method, it was found that selected epitopes in the tryptic ß-LG peptides remain stable over the ripening period of the cheese. For four of the tryptic ß-LG peptides detected by immunostaining, the amino acid sequence was identified using MALDI-TOF-MS/MS. One of the peptides identified was the semi-tryptic peptide VYVEELKPTP. A linear relationship was found between the content of this peptide in cheese and the proportion of high-heated milk in the cheese milk. ß-LG enrichment factors of 1.72 (n = 3, sample set I) and 1.33 ± 0.19 (n = 1, sample set II) were determined for the cheese samples containing 30% high-heated milk compared to the non-enriched samples. The relative ß-LG contents in the cheese samples with 30% high-heated milk were calculated to be 4.35% ± 0.39% (sample set I) and 9.11% ± 0.29% (sample set II) using a one-point calibration. It can be concluded that the HPTLC-IS method used is a suitable tool for the analysis of whey protein accumulation in cheese, being therefore potentially directly applicable on an industrial scale. For more accurate quantification of the whey protein content in cheese, an enhanced calibration curve needs to be applied.

Characterization of Conjugates between α-Lactalbumin and Benzyl Isothiocyanate-Effects on Molecular Structure and Proteolytic Stability.

In complex foods, bioactive secondary plant metabolites (SPM) can bind to food proteins. Especially when being covalently bound, such modifications can alter the structure and, thus, the functional and biological properties of the proteins. Additionally, the bioactivity of the SPM can be affected as well. Consequently, knowledge of the influence of chemical modifications on these properties is particularly important for food processing, food safety, and nutritional physiology. As a model, the molecular structure of conjugates between the bioactive metabolite benzyl isothiocyanate (BITC, a hydrolysis product of the glucosinolate glucotropaeolin) and the whey protein α-lactalbumin (α-LA) was investigated using circular dichroism spectroscopy, anilino-1-naphthalenesulfonic acid fluorescence, and dynamic light scattering. Free amino groups were determined before and after the BITC conjugation. Finally, mass spectrometric analysis of the BITC-α-LA protein hydrolysates was performed. As a result of the chemical modifications, a change in the secondary structure of α-LA and an increase in surface hydrophobicity and hydrodynamic radii were documented. BITC modification at the ε-amino group of certain lysine side chains inhibited tryptic hydrolysis. Furthermore, two BITC-modified amino acids were identified, located at two lysine side chains (K32 and K113) in the amino acid sequence of α-LA.

Benzyl isothiocyanate-modified α-lactalbumin - Two-dimensional high-performance thin-layer chromatography for analyzing modified peptides.

In complex food matrices, non-directed reactions between food proteins and secondary plant metabolites (SPM) are conceivable. In this study, the interaction between the bioactive metabolite from garden cress (Lepidium sativum) and selected Brassicaceae - benzyl isothiocyanate (BITC) - and the dairy protein α-lactalbumin (α-LA) was investigated. It was focused on monitoring the proteolytic degradation behaviour of unmodified and BITC-modified α-LA with two-dimensional high-performance thin-layer chromatography (2D-HPTLC). The two-dimensional approach of HPTLC offers high resolution in the separation of complex peptide mixtures and might enable differentiation of protein modifications. Based on the specific peptide patterns of native and modified peptides, conclusions can be drawn about differences in protein/peptide polarity, location of a modification, and digestibility. The aim was to characterize tryptically hydrolyzed unmodified and BITC-modified peptides using the 2D method and to investigate the influence of BITC modification of α-LA on polarity and digestibility. To determine the repeatability of peptide separation by 2D-HPTLC, the unmodified and BITC-modified protein hydrolyzates were separated six times. The absolute standard deviations between the retardation factors of the individual peptide spots varied between 0.52 and 4.79 mm for the x-coordinates and between 0.41 and 6.47 mm for the y-coordinates for all three samples. Here, the mean relative standard deviations ranged from 5.80 to 10.4% for the x-coordinates and from 5.91 to 18.3% for the y-coordinates. The results of the tryptic hydrolysis indicated that, depending on the concentration of BITC used, the modification sterically hinders the cleavage sites for the enzyme, resulting in a reduced digestibility. Covalent binding of the hydrophobic BITC altered the digestibility and polarity of the protein, leading to a difference in peptide patterns between the unmodified and modified α-LA. It was concluded that the reaction was undirected, resulting in a mixture of unmodified and modified peptides, and that elongated modified peptides were formed by BITC blocking of trypsin cleavage sites.

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS.

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0-15 kDa, 15-35 kDa, 35-70 kDa and 70-250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.

Electroblotting through Enzymatic Membranes to Enhance Molecular Tissue Imaging.

MALDI-TOF mass spectrometry imaging (MSI) is a powerful tool for studying biomolecule localization in tissue. Protein distributions in tissue provide important histological information; however, large proteins exhibit a high limit of detection in MALDI-MS when compared to their corresponding smaller proteolytic peptides. As a result, several techniques have emerged to digest proteins into more detectable peptides for imaging. Digestion is typically accomplished through trypsin deposition on the tissue, but this technique increases the complexity of the tissue microenvironment, which can limit the number of detectable species. This proof-of-principle study explores tryptic tissue digestion during electroblotting through a trypsin-containing membrane. This approach actively extracts and enzymatically digests proteins from mouse brain tissue sections while simultaneously reducing the complexity of the tissue microenvironment (compared to trypsin deposition on the surface) to obtain an increased number of detectable peptide fragments. The method does not greatly compromise spatial location or require expensive devices to uniformly deposit trypsin on tissue. Using electrodigestion through membranes, we detected and tentatively identified several tryptic peptides that were not observed after on-tissue digestion. Moreover, the use of pepsin rather than trypsin in digestion membranes allows extraction and digestion at low pH to detect peptides from a complementary subset of tissue proteins. Future studies will aim to further improve the method, including changing the substrate membrane to increase spatial resolution and the number of detected peptides.

Human Blood Plasma Investigation Employing 2D UPLC-UDMSE Data-Independent Acquisition Proteomics.

Proteomic tools are especially useful when it comes to investigating complex samples such as human blood plasma, in which protein quantities can span across up to ten orders of magnitude. Ultra definition mass spectrometry, in combination with two-dimensional liquid chromatography, provides better coverage of complex proteomes and allows for better control of collision energy, keeping the fragmentation benefits of high collision energy associated with drift time measurements from ion mobility separation. Here, we present a protocol to assist in the identification of proteins in human blood plasma and other similar samples with a large dynamic range.

Revealing the changes of IgG subclass-specific N-glycosylation in colorectal cancer progression by high-throughput assay.

PURPOSE: The changes of glycosylation of different IgG subclass in colorectal cancer (CRC) were rarely investigated. The authors aimed to use a simple and high-throughput analytical method to explore the changes of subclass-specific IgG glycosylation in CRC, and to find the specific glyco-biomarkers for early detection of this disease. EXPERIMENTAL DESIGN: Serum samples from 71 cancer patients and 22 benign patients with 50 age- and sex-matched healthy controls were collected from two independent cohorts. Subclass-specific IgG glycosylation was profiled by MALDI-MS followed by the structural identification through MALDI-MS/MS. The exported MS data was automatically and rapidly processed by the self-developed MATLAB code. RESULTS: Statistical analysis suggested the significantly decreased galactosylation and remarkably increased agalactosylation of IgG1 or IgG2 in the malignant transformation of CRC, which enables the differentiation between cancer patients and healthy controls. The changes of glycan features were elucidated by the exploration of individual glycopeptides, showing the biantennary fucosylated glycan without galactose (H3N4F1) or with two galactose (H5N4F1) of IgG1 and IgG2 could distinguish cancer group from both benign and control groups. CONCLUSIONS AND CLINICAL RELEVANCE: Through the simple and high-throughput procedures, this study revealed the important role of IgG glycopeptides in the premature pathology of CRC.

A novel method for large-scale confirmation of protein structures and surface accessible modification sites.

Protein function is modulated via different levels including their structure and chemical modifications, but method for monitoring of protein structures and their modifications on a large scale is far from mature. In this study, we present a shot-gun proteomic method, which combine ultrafiltration with limited tryptic proteolysis (FLiP) to enrich the surface accessible peptides and modification sites on protein structures. FLiP enable high throughput confirmation of the structural information of 1939 proteins. Based on this, we identified 120 types of modifications located on the surface accessible regions of proteins, including some rare modification types like phosphorylation of asparagine and acetylation of threonine. Our data provides a comprehensive picture of the spatial distribution of multiple modification types on protein structures. Collectively, our work provides a promising tool for large-scale confirmation of protein structures and unbiased discovery of multiple proteins surface assessable post-translational modifications, which may also benefits the study of rare modification types.

Simultaneous quantification of major capsid protein of human papillomavirus 16 and human papillomavirus 18 in multivalent human papillomavirus vaccines by liquid chromatography-tandem mass spectrometry.

Human papillomavirus (HPV) vaccination is the most effective mean to prevent HPV infection and cervical carcinoma. Licensed HPV prophylactic vaccines are formulated to contain a defined amount of different major capsid protein (L1), the critical antigen to elicit protection. No method is currently available to simultaneously quantify individual L1s in multivalent vaccines, presenting a daunting challenge for the quality control of HPV vaccines. Here, HPV16 and HPV18 L1 can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using tryptic digestion without pre-digestion reduction and alkylation in multiple reaction monitoring (MRM) mode. Two signature peptides were selected to be the markers of the two L1s and can be well separated within 5.1 min. Their linear calibration curves were both obtained in the range of 20-500 nmol/L (R2 > 0.990). To HPV16 L1, intra/inter assay precisions and accuracies of the assay were below 11% and between 83.96-113.57%. While for HPV18 L1, they were below 12% and between 81.40-103.49%. In addition, the limits of quantitation (LOQ) were as low as 2.8 nmol/L for HPV16 L1 and 1.7 nmol/L for HPV18 L1, respectively, representing about 68 and 112 times more sensitive than those obtained with Smith Bicinchoninic Acid (BCA) assay. This LC-MS/MS method can be applied to the quantification of both bulk products and the final multivalent vaccines. This method is superior to the current assays in terms of sensitivity, specificity, precision, accuracy and throughput; it could become the method of choice for absolute quantification of proteins in multivalent vaccines.

A concept to make low-abundance endogenous renin accessible to mass spectrometry: A multistep experimental design approach.

Renin is the rate-limiting step within the renin-angiotensin-aldosterone system, but the reliable quantification of human endogenous renin levels by liquid chromatography coupled with mass spectrometry remains challenging. The complex sample matrix triggering ion suppression and the detection of the low-abundance as well as the proteolytical-resistant renin make a hybrid approach using immunocapture coupled with LC-HRMS a promising method for investigation. Therefore, in-silico digestion and BLAST® experiments were conducted in order to identify the unique amino acid sequence for mass spectrometric detection. To enhance mass spectrometric response, impacting parameters within the denaturation, alkylation, and digestion experiments were identified and optimized by a multistep Design of Experiments process. The optimal denaturation buffer consisted of RapiGest® and urea, leading to a signature peptide intensity increase of 56% at 20 °C, whereas the optimal reducing agent improved intensity by 27%. The most effective generation of signature peptide I was achieved using a high trypsin concentration and a low incubation temperature enhancing digestion by 75%. The applicability of this hybrid approach was confirmed in human matrix and allowed for a fivefold reduction in total assay procedure time without limiting the reliable quantification if compared to a conventional digestion approach.

A recommended and verified procedure for in situ tryptic digestion of formalin-fixed paraffin-embedded tissues for analysis by matrix-assisted laser desorption/ionization imaging mass spectrometry.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin-fixed paraffin-embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross-links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.

Liquid chromatography-tandem mass spectrometry of recombinant human extracellular superoxide dismutase (rhSOD3) in mouse plasma and its application to pharmacokinetic study.

The antioxidant enzyme human extracellular superoxide dismutase (SOD3) is a promising biopharmaceutical candidate for the treatment of various diseases. To support the early development of SOD3 as a biopharmaceutical, a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry procedure was developed and validated for the determination of SOD3 levels in the plasma of ICR mice. After purification with Ni-NTA magnetic beads and digestion with trypsin, SOD3 signature peptides and internal standard signature peptide (ISP) were separated via high performance liquid chromatography using a Zorbax C18 column (2.1 × 50 mm, 3.5 µm) and a mobile phase consisting of 10 mM ammonium formate, 0.1% formic acid, and acetonitrile. The analyte and ISP were detected via a tandem mass spectrometer in electrospray ionization and multiple reaction monitoring modes to select both the signature peptide for SOD3 at m/z 669 to 969 and the ISP at m/z 655 to 941 in the positive ion mode. The calibration curves were linear (r > 0.99) between 5 and 1000 µg/mL with a lower limit of quantification of 5 µg/mL. The relative standard deviation ranged from 3.08 to 8.84% while the relative error ranged from -0.13 to -9.56%. This method was successfully applied to a preclinical pharmacokinetic study of SOD3 in male ICR mice.

Determination of food allergens by LC-MS: Impacts of sample preparation, food matrix, and thermal processing on peptide detectability and quantification.

Food allergies are a growing worldwide concern and the contamination of products with food allergens represents a significant health risk to allergic consumers. With the introduction of reference doses, quantitative methods are needed for the monitoring of allergen levels, and the potential of LC-MS/MS is of hugely growing interest. In this study, we demonstrate that relevant food matrices (bakery products and chocolates) and thermal food processing substantially influence the quantification of 18 marker peptides from various nut and peanut allergens via targeted proteomics. In addition, we characterize the individual release kinetics of marker peptides and provide examples for metastable marker peptide candidates. Matrix recovery rates overall ranged between 15 and 250% with the observed variation being linked to the individual peptide structure as well as to specific matrix interferences. In contrast, thermal processing considerably influences the detectability of allergens on the protein level as different marker peptides from the identical parent allergen are similarly affected, leading to a loss in signal of up to 83% in extreme cases after a 45-min simulated baking. Provided data are finally used for evaluation of different calibrators as well as the overall potential and challenges of LC-MS for the absolute quantification of food allergens. SIGNIFICANCE: With the scientific discussion moving towards a risk-based management of food allergens, including the establishment of threshold doses, robust methods for the absolute quantification of allergens in food samples are urgently needed. Because the currently used antibody- and DNA-based technologies show severe limitations in terms of specificity and reproducibility, LC-MS has emerged as a promising alternative. Its application to absolute quantification, however, first requires an understanding of the various impacts that affect quantification results, including different food matrices, sample preparation, and thermal processing of foodstuffs. Knowledge of these factors, which are assessed as part of a comprehensive survey in this study, is also an important prerequisite to evaluate means of calibration for an LC-MS-based quantification of food allergens.