Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer's Disease.

To elucidate the role of Tau isoforms and post-translational modification (PTM) stoichiometry in Alzheimer's disease (AD), we generated a high-resolution quantitative proteomics map of 95 PTMs on multiple isoforms of Tau isolated from postmortem human tissue from 49 AD and 42 control subjects. Although Tau PTM maps reveal heterogeneity across subjects, a subset of PTMs display high occupancy and frequency for AD, suggesting importance in disease. Unsupervised analyses indicate that PTMs occur in an ordered manner, leading to Tau aggregation. The processive addition and minimal set of PTMs associated with seeding activity was further defined by analysis of size-fractionated Tau. To summarize, features in the Tau protein critical for disease intervention at different stages of disease are identified, including enrichment of 0N and 4R isoforms, underrepresentation of the C terminus, an increase in negative charge in the proline-rich region (PRR), and a decrease in positive charge in the microtubule binding domain (MBD).

Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis.

Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.

Benchmarking digital PCR partition classification methods with empirical and simulated duplex data.

Digital PCR (dPCR) is a highly accurate technique for the quantification of target nucleic acid(s). It has shown great potential in clinical applications, like tumor liquid biopsy and validation of biomarkers. Accurate classification of partitions based on end-point fluorescence intensities is crucial to avoid biased estimators of the concentration of the target molecules. We have evaluated many clustering methods, from general-purpose methods to specific methods for dPCR and flowcytometry, on both simulated and real-life data. Clustering method performance was evaluated by simulating various scenarios. Based on our extensive comparison of clustering methods, we describe the limits of these methods, and formulate guidelines for choosing an appropriate method. In addition, we have developed a novel method for simulating realistic dPCR data. The method is based on a mixture distribution of a Poisson point process and a skew-$t$ distribution, which enables the generation of irregularities of cluster shapes and randomness of partitions between clusters ('rain') as commonly observed in dPCR data. Users can fine-tune the model parameters and generate labeled datasets, using their own data as a template. Besides, the database of experimental dPCR data augmented with the labeled simulated data can serve as training and testing data for new clustering methods. The simulation method is available as an R Shiny app.

Dynamic changes of gut microbiota in mouse models of metabolic dysfunction-associated steatohepatitis and its transition to hepatocellular carcinoma.

Dysbiosis of gut microbiota may account for pathobiology in simple fatty liver (SFL), metabolic dysfunction-associated steatohepatitis (MASH), fibrotic progression, and transformation to MASH-associated hepatocellular carcinoma (MASH-HCC). The aim of the present study is to investigate gut dysbiosis in this progression. Fecal microbial rRNA-16S sequencing, absolute quantification, histopathologic, and biochemical tests were performed in mice fed high fat/calorie diet plus high fructose and glucose in drinking water (HFCD-HF/G) or control diet (CD) for 2, 16 weeks, or 14 months. Histopathologic examination verified an early stage of SFL, MASH, fibrotic, or MASH-HCC progression with disturbance of lipid metabolism, liver injury, and impaired gut mucosal barrier as indicated by loss of occludin in ileum mucosa. Gut dysbiosis occurred as early as 2 weeks with reduced α diversity, expansion of Kineothrix, Lactococcus, Akkermansia; and shrinkage in Bifidobacterium, Lactobacillus, etc., at a genus level. Dysbiosis was found as early as MAHS initiation, and was much more profound through the MASH-fibrotic and oncogenic progression. Moreover, the expansion of specific species, such as Lactobacillus johnsonii and Kineothrix alysoides, was confirmed by an optimized method for absolute quantification. Dynamic alterations of gut microbiota were characterized in three stages of early SFL, MASH, and its HCC transformation. The findings suggest that the extent of dysbiosis was accompanied with MASH progression and its transformation to HCC, and the shrinking or emerging of specific microbial species may account at least in part for pathologic, metabolic, and immunologic alterations in fibrogenic progression and malignant transition in the liver.

Quantification of snake venom proteomes by mass spectrometry-considerations and perspectives.

The advent of soft ionization mass spectrometry-based proteomics in the 1990s led to the development of a new dimension in biology that conceptually allows for the integral analysis of whole proteomes. This transition from a reductionist to a global-integrative approach is conditioned to the capability of proteomic platforms to generate and analyze complete qualitative and quantitative proteomics data. Paradoxically, the underlying analytical technique, molecular mass spectrometry, is inherently nonquantitative. The turn of the century witnessed the development of analytical strategies to endow proteomics with the ability to quantify proteomes of model organisms in the sense of "an organism for which comprehensive molecular (genomic and/or transcriptomic) resources are available." This essay presents an overview of the strategies and the lights and shadows of the most popular quantification methods highlighting the common misuse of label-free approaches developed for model species' when applied to quantify the individual components of proteomes of nonmodel species (In this essay we use the term "non-model" organisms for species lacking comprehensive molecular (genomic and/or transcriptomic) resources, a circumstance that, as we detail in this review-essay, conditions the quantification of their proteomes.). We also point out the opportunity of combining elemental and molecular mass spectrometry systems into a hybrid instrumental configuration for the parallel identification and absolute quantification of venom proteomes. The successful application of this novel mass spectrometry configuration in snake venomics represents a proof-of-concept for a broader and more routine application of hybrid elemental/molecular mass spectrometry setups in other areas of the proteomics field, such as phosphoproteomics, metallomics, and in general in any biological process where a heteroatom (i.e., any atom other than C, H, O, N) forms integral part of its mechanism.

Highly sensitive therapeutic drug monitoring of infliximab in serum by targeted mass spectrometry in comparison to ELISA data.

BACKGROUND: Presently, antibody concentration measurements for patients undergoing treatment are predominantly determined by ELISA, which still comes with known disadvantages. Therefore, our aim was to establish a targeted mass-spectrometric assay enabling the reproducible absolute quantification of peptides from the hypervariable and interaction regions of infliximab. METHODS: Peptides of infliximab were measured post-trypsin digestion and subsequent separation on a Vanquish Horizon UHPLC coupled to a TSQ Altis Triple-Quad mass spectrometer. Normalization and absolute quantification were conducted using stable isotope-synthesized peptides. Calibration curves covering a range of 0.25-50 µg/ml were employed for quantitation. RESULTS: We demonstrated the substantial influence of peptide selection, choice of hydrolase for digestion, and digestion time on absolute peptide yield (28-44% for peptide 1 and 64-97% for peptide 2). Furthermore, we showed that the generated calibration curves for absolute quantification were highly reproducible and robust (LLOQ1 0.72 µg/ml and LLOQ2 1.00 µg/ml) over several months. In comparison to ELISA values, the absolute values obtained by mass spectrometry often yielded lower results for both targeted peptides. CONCLUSIONS: In this study, a semi-automated workflow was employed and tested with 8 patients and corresponding replicates (n = 3-4). We demonstrated the robust implementation of calibration curves for the absolute quantification of infliximab in patient samples, with coefficients of variation ranging from 0.5 to 9%. Taken together, we have developed a platform enabling the rapid (2 days of sample preparation and 30 min of measurement time per sample) and robust quantification of Infliximab antibody concentration in patients. The use of mass spectrometry also facilitates the straightforward expansion of the method to include additional antibody peptides.

Reproducibility of absolute quantification of adenosine triphosphate and inorganic phosphate in the liver with localized 31P-magnetic resonance spectroscopy at 3-T using different coils.

Concentrations of the key metabolites of hepatic energy metabolism, adenosine triphosphate (ATP) and inorganic phosphate (Pi), can be altered in metabolic disorders such as diabetes mellitus. 31Phosphorus (31P)-magnetic resonance spectroscopy (MRS) is used to noninvasively measure hepatic metabolites, but measuring their absolute molar concentrations remains challenging. This study employed a 31P-MRS method based on the phantom replacement technique for quantifying hepatic 31P-metabolites on a 3-T clinical scanner. Two surface coils with different size and geometry were used to check for consistency in terms of repeatability and reproducibility and absolute concentrations of metabolites. Day-to-day (n = 8) and intra-day (n = 6) reproducibility was tested in healthy volunteers. In the day-to-day study, mean absolute concentrations of γ-ATP and Pi were 2.32 ± 0.24 and 1.73 ± 0.26 mM (coefficient of variation [CV]: 7.3% and 8.8%) for the single loop, and 2.32 ± 0.42 and 1.73 ± 0.27 mM (CVs 6.7% and 10.6%) for the quadrature coil, respectively. The intra-day study reproducibility using the quadrature coil yielded CVs of 4.7% and 6.8% for γ-ATP and Pi without repositioning, and 6.3% and 7.1% with full repositioning of the volunteer. The results of the day-to-day data did not differ between coils and visits. Both coils robustly yielded similar results for absolute concentrations of hepatic 31P-metabolites. The current method, applied with two different surface coils, can be readily utilized in long-term and interventional studies. In comparison with the single loop coil, the quadrature coil also allows measurements at a greater distance between the coil and liver, which is relevant for studying people with obesity.

Transporter Protein Expression of Corneal Epithelium in Rabbit and Porcine: Evaluation of Models for Ocular Drug Transport Study.

The transcorneal route is the main entry route for drugs to the intraocular parts, after topical administration. The outer surface, the corneal epithelium (CE), forms the rate-limiting barrier for drug permeability. Information about the role and protein expression of drug and amino acid transporter proteins in the CE is sparse and lacking. The aim of our study was to characterize transporter protein expression in rabbit and porcine CE to better understand potential drug and nutrient absorption after topical administration. Proteins, mainly Abc and Slc transporters, were characterized with quantitative targeted absolute proteomics and global untargeted proteomics methods. In the rabbit CE, 24 of 48 proteins were detected in the targeted approach, and 21 of these were quantified. In the porcine CE, 26 of 58 proteins were detected in the targeted approach, and 20 of these were quantified. Among these, 15 proteins were quantified in both animals: 4f2hc (Slc3a2), Aqp0, Asct1 (Slc1a4), Asct2 (Slc1a5), Glut1 (Slc2a1), Hmit (Slc2a13), Insr, Lat1 (Slc7a5), Mct1 (Slc16a1), Mct2 (Slc16a7), Mct4 (Slc16a3), Mrp 4 (Abcc4), Na+/K+-ATPase, Oatp3a1 (Slco3a1), and Snat2 (Slc38a2). Overall, the global proteomics results supported the targeted proteomics results. Organic anion transporting polypeptide Oatp3a1 was detected and quantified for the first time in both rabbit (1.4 ± 0.4 fmol/cm2) and porcine (11.1 ± 5.3 fmol/cm2) CE. High expression levels were observed for L-type amino acid transporter, Lat1, which was quantified with newly selected extracellular domain peptides in rabbit (48.9 ± 11.8 fmol/cm2) and porcine (37.6 ± 11.5 fmol/cm2) CE. The knowledge of transporter protein expression in ocular barriers is a key factor in the successful design of new ocular drugs, pharmacokinetic modeling, understanding ocular diseases, and the translation to human.

Identification and optimization of parameters for accurate quantification of RNA by RT-dPCR.

Reverse transcription-digital PCR (RT-dPCR) is attracting attention as a method that enables SI-traceable RNA quantification without calibration, but its accuracy and bias have not been thoroughly studied. In this study, the accurate quantification of RNA by the RT-dPCR method was investigated using NMIJ CRM 6204-b, an RNA certified reference material whose certified value was assigned by orthogonal chemical measurement methods. Moreover, a two-step RT-dPCR method was adopted to examine in detail the conditions for the RT reaction process, which was expected to be the major uncertainty component in the RT-dPCR measurement. Optimization experiments revealed that the type of reverse transcriptase, the concentration of template RNA, and the type and concentration of primers in the RT reaction affected the value quantified by RT-dPCR. Under the optimal conditions, the value quantified by RT-dPCR, 76.4 ng/µL ± 6.7 ng/µï»¿L (the quantified value ± expanded uncertainty (k = 2)), was consistent with the certified value, 68.2 ng/µï»¿L ± 5.8 ng/µï»¿L, of NMIJ CRM 6204-b RNA 1000-A within the expanded uncertainty. From the results of the uncertainty evaluation, the relative combined uncertainty of the RT-dPCR method was 4.42%, and the major uncertainty components in the RT-dPCR method were the preparation of RT solution (3.68%), the inter-day difference (1.80%), and the RT reaction (1.30%). Together, the results suggested that the contribution of the RT reaction process to the total uncertainty was greater than that of the dPCR process.

Monitoring the Photorhabdus spp. bacterial load in Heterorhabditis bacteriophora dauer juveniles over different storage times and temperatures: A molecular approach.

Biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora can vary in virulence (quality). The influence of their symbiotic bacteria Photorhabdus spp. inside the infective dauer juvenile (DJ) on DJ quality has not received much attention in the past. The presence of the bacteria in the DJ is crucial for its biocontrol potential. This investigation provides a method to quantify the bacterial load inside the DJ based on a qPCR technique. Information from the genome of Photorhabdus laumondii strain DE2 was used to identify single copy genes with no homology to any other bacterial accessions. One gene (hereby named CG2) was selected for primers design and for further qPCR experiments. Cross-amplification tests with P. thracensis and P. kayaii, also symbionts of H. bacteriophora, were positive, whereas no amplicons were produced for P. temperata or Xenorhabdus nematophila. We tested our qPCR system in DJ populations carrying defined proportions of bacteria-free (axenic) vs bacteria-carrying nematodes. With an increasing proportion of axenic DJ in a population, virulence declined, and the virulence was proportional to the amount of bacterial DNA detected in the population by qPCR. Along liquid storage over long time, virulence also decreased, and this factor correlated with the reduction of bacterial DNA on the respective DJ population. We observed that stored DJ kept virulent up to 90 days and thereafter the virulence as well as the amount of bacterial DNA drastically decreased. Storage temperature also influenced the bacterial survival. Inside formulated DJ, the loss of bacterial DNA on the DJ population was accelerated under storage temperatures below 7.5 °C, suggesting that reproduction of the bacterial cells takes place when growth temperature is favorable. The role of bacterial survival inside stored DJ can now be adequately addressed using this molecular quality-control technique.

Eye proteome of Drosophila melanogaster.

Drosophila melanogaster is a popular model organism to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies, aging, light-induced damage, or dietary deficiencies. Large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which led to the discovery of key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins, including the absolute (molar) quantities of 43 proteins in the eye of adult male Drosophila reared on standard laboratory food. This work provides a generic and expandable resource for further genetic, pharmacological, and dietary studies.

Change of histone H3 lysine 14 acetylation stoichiometry in human monocyte derived macrophages as determined by MS-based absolute targeted quantitative proteomic approach: HIV infection and methamphetamine exposure.

BACKGROUND: Histones posttranslational modification represent an epigenetic mechanism that regulate gene expression and other cellular processes. Quantitative mass spectrometry used for the absolute quantification of such modifications provides further insight into cellular responses to extracellular insults such as infections or toxins. Methamphetamine (Meth), a drug of abuse, is affecting the overall function of the immune system. In this report, we developed, validated and applied a targeted, MS-based quantification assay to measure changes in histone H3 lysine 14 acetylation (H3K14Ac) during exposure of human primary macrophages to HIV-1 infection and/or Meth. METHODS: The quantification assay was developed and validated to determine H3K14Ac stoichiometry in histones that were isolated from the nuclei of control (CIC) and exposed to Meth before (CIM) or/and after (MIM) HIV-infection human monocyte-derived macrophages (hMDM) of six donors. It was based on LC-MS/MS measurement using multiple reaction monitoring (MRM) acquisition of the unmodified and acetylated form of lysine K14 of histone H3 9KSTGGKAPR17 peptides and the corresponding stable isotope labeled (SIL) heavy peptide standards of the same sequences. The histone samples were propionylated (Poy) pre- and post- trypsin digestion so that the sequences of the monitored peptides were: K[Poy]STGGK[1Ac]APR, K[Poy]STGGK[1Ac]APR-heavy, K[Poy]STGGK[Poy]APR and K[Poy]STGGK[Poy]APR-heavy. The absolute amounts of the acetylated and unmodified peptides were determined by comparing to the abundances of their SIL standards, that were added to the samples in the known concentrations, and, then used for calculation of H3K14Ac stoichiometry in CIC, CIM and MIM hMDM. RESULTS: The assay was characterized by LLOD of 0.106 fmol/µL and 0.204 fmol/µL for unmodified and acetylated H3 9KSTGGKAPR17 peptides, respectively. The LLOQ was 0.5 fmol/µL and the linear range of the assay was from 0.5 to 2500 fmol/µL. The absolute abundances of the quantified peptides varied between the donors and conditions, and so did the H3K14Ac stoichiometry. This was rather attributed to the samples nature itself, as the variability of their triplicate measurements was low. CONCLUSIONS: The developed LC-MS/MS assay enabled absolute quantification of H3K14Ac in exposed to Meth HIV-infected hMDM. It can be further applied determination of this PTM stoichiometry in other studies on human primary macrophages.

Fluorescence-based absolute quantification of near-infrared probes in tissue extracts for biodistribution analyses.

In recent years, significant progress has been made in the development of fluorescent contrast agents for clinical applications. For the development of a fluorescent probe, it is crucial to evaluate its safety profile, including biodistribution. Specific methods need to be developed for the absolute quantification of fluorescent probes in tissue specimens from animals administered with test compounds in the framework of biodistribution/efficacy/toxicity studies. Here, we describe a new method for the absolute quantification of fluorescent probes in tissue specimens from animals administered with compounds that have absorption and emission wavelength in the Near-Infrared region (600-800 nm). The protocol is based on the standard addition approach in order to minimize the interference of the matrix on the analyte signal causing inaccuracy in the absolute determination of the concentration. The measurement of the fluorescence intensity is done via a microplate reader. The method has been fully validated and applied for the quantification of a fluorescence-guided surgery targeted contrast agent in a Good Laboratory Practice (GLP) biodistribution study. Results clearly demonstrate that this procedure is fully applicable in a preclinical setting and that it overcomes common issues associated with fluorescence signal quantification in tissue extracts.

Digital CRISPR Systems for the Next Generation of Nucleic Acid Quantification.

Digital CRISPR (dCRISPR) assays are an emerging platform of molecular diagnostics. Digital platforms introduce absolute quantification and increased sensitivity to bulk CRISPR assays. With ultra-specific targeting, isothermal operation, and rapid detection, dCRISPR systems are well-prepared to lead the field of molecular diagnostics. Here we summarized the common Cas proteins used in CRISPR detection assays. The methods of digital detection and critical performance factors are examined. We formed three strategies to frame the landscape of dCRISPR systems: (1) amplification free, (2) in-partition amplification, and (3) two-stage amplification. We also compared the performance of all systems through the limit of detection (LOD), testing time, and figure of merit (FOM). This work summarizes the details of digital CRISPR platforms to guide future development. We envision that improvements to LOD and dynamic range will position dCRISPR as the leading platform for the next generation of molecular biosensing.

High-Throughput Absolute Quantification Sequencing Reveals that a Combination of Leguminous Shrubs Is Effective in Driving Soil Bacterial Diversity During the Process of Desertification Reversal.

Desertification leads to the extreme fragility of ecosystems and seriously threatens ecosystem functioning in desert areas. The planting of xerophytes, especially leguminous shrubs, is an effective and common means to reverse desertification. Soil microorganisms play a crucial role in nutrient cycling and energy flow in ecosystems. However, the effects of introducing leguminous shrubs on soil microbial diversity and the relevant mechanisms are not clear. Here, we employed the high-throughput absolute quantification 16S rRNA sequencing method to analyze the diversity of soil bacteria in sand-fixing areas of mixed shrublands with three combinations of shrubs, i.e., C. korshinskii × Corethrodendron scoparium (CaKCoS), C. korshinskii × Calligonum mongolicum (CaKCaM), and C. scoparium × C. mongolicum (CoSCaM), in the south of the Mu Us Sandy Land, China. This area suffered from moving dunes 20 years ago, but after introducing these shrubs to fix the dunes, the ecosystem was restored. Additionally, the effects of soil physicochemical properties on soil bacterial composition and diversity were analyzed with redundancy analysis (RDA) and structural equation modeling (SEM). It was found that the Shannon index of soil bacteria in CaKCoS was significantly higher than that in CaKCaM and CoSCaM, and the abundance of the dominant phyla, including Actinobacteria, Proteobacteria, Acidobacteria, Chloroflexi, Planctomycetes, Thaumarchaeota, Armatimonadetes, candidate_division_WPS-1, and Nitrospirae, increased significantly in CaKCoS and CaKCaM compared to that in CoSCaM. RDA showed that the majority of soil properties, such as total nitrogen (TN), available potassium (AK), N:P ratio, soil moisture (SM), and available phosphorus (AP), were important soil environmental factors affecting the abundance of the dominant phyla, and RDA1 and RDA2 accounted for 56.66% and 2.35% of the total variation, respectively. SEM showed that the soil bacterial α-diversity was positively affected by the soil organic carbon (SOC), N:P ratio, and total phosphorus (TP). Moreover, CaKCoS had higher SM, total carbon (TC), total potassium (TK), and AP than CaKCaM and CoSCaM. Collectively, these results highlight a conceptual framework in which the combination of leguminous shrubs can effectively drive soil bacterial diversity by improving soil physicochemical properties and maintaining ecosystem functioning during desertification reversal.

Quantification of arbuscular mycorrhizal fungi root colonization in wheat, tomato, and leek using absolute qPCR.

Arbuscular mycorrhizal fungi (AMF) form symbioses with most terrestrial plants and are known to have a positive effect on plant growth and health. Different methodologies have been developed to assess the AMF-plant symbiosis. The most applied method, which involves staining of roots and microscopic observation of the AMF structures, is tedious and time-consuming and the results are highly dependent on the observer. Using quantitative polymerase chain reaction (qPCR) to quantify AMF root colonization represents a reliable, high-throughput technique that allows the assessment of numerous samples. Quantification with qPCR can be performed through two methods: relative quantification and absolute quantification. In relative quantification, the target gene is normalized with a reference gene. On the other hand, absolute quantification involves the use of a standard curve, for which template DNA is serially diluted. In a previous paper, we validated the primer pair AMG1F and AM1 for a relative quantification approach to assess AMF root colonization in Petunia. Here, we tested the same primers with an absolute quantification approach and compared the results with the traditional microscopy method. We evaluated the qPCR method with three different crops, namely, wheat (cv. Colmetta and Wiwa), tomato, and leek. We observed a strong correlation between microscopy and qPCR for Colmetta (r = 0.90, p < 0.001), Wiwa (r = 0.94, p < 0.001), and tomato (r = 0.93, p < 0.001), but no correlation for leek (r = 0.27, p = 0.268). This highlights the importance of testing the primer pair for each specific crop.

Median-Based Absolute Quantification of Proteins Using Fully Unlabeled Generic Internal Standard (FUGIS).

By reporting the molar abundance of proteins, absolute quantification determines their stoichiometry in complexes, pathways, or networks. Typically, absolute quantification relies either on protein-specific isotopically labeled peptide standards or on a semiempirical calibration against the average abundance of peptides chosen from arbitrarily selected proteins. In contrast, a generic protein standard FUGIS (fully unlabeled generic internal standard) requires no isotopic labeling, chemical synthesis, or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of the peptide peaks produced by the tryptic digestion of FUGIS is used as a single-point calibrant to determine the molar abundance of any codigested protein. Powered by FUGIS, median-based absolute quantification (MBAQ) outperformed other methods of untargeted proteome-wide absolute quantification.

Addressing the Protease Bias in Quantitative Proteomics.

Protein quantification strategies using multiple proteases have been shown to deliver poor interprotease accuracy in label-free mass spectrometry experiments. By utilizing six different proteases with different cleavage sites, this study explores the protease bias and its effect on accuracy and precision by using recombinant protein standards. We established 557 SRM assays, using a recombinant protein standard resource, toward 10 proteins in human plasma and determined their concentration with multiple proteases. The quantified peptides of these plasma proteins spanned 3 orders of magnitude (0.02-70 µM). In total, 60 peptides were used for absolute quantification and the majority of the peptides showed high robustness. The retained reproducibility was achieved by quantifying plasma proteins using spiked stable isotope standard recombinant proteins in a targeted proteomics workflow.

Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants.

We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by Staphylococcus aureus and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.

FastCAT Accelerates Absolute Quantification of Proteins Using Multiple Short Nonpurified Chimeric Standards.

Absolute (molar) quantification of clinically relevant proteins determines their reference values in liquid and solid biopsies. The FastCAT (for Fast-track QconCAT) method employs multiple short (<50 kDa), stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q)-peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R)-peptides that relate its abundance to a single protein standard (bovine serum albumin, BSA). FastCAT not only alleviates the need to purify CP or use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but also improves the accuracy, precision, and dynamic range of the absolute quantification by grouping Q-peptides according to the expected abundance of the target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantification of neurological markers in human cerebrospinal fluid at the low ng/mL level.