Stable Isotope-labeled Peptides Research Articles

Differential tissue abundance of membrane-bound drug metabolizing enzymes and transporter proteins by global proteomics.

Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are critical for scaling in vitro and animal data to humans for accurate prediction and interpretation of drug clearance and toxicity. Targeted DMET proteomics which relies on synthetic stable isotope-labeled surrogate peptides as calibrators, is routinely used for the quantification of selected proteins; however, the technique is limited to the quantification of a small number of proteins. Although the global proteomics-based total protein approach (TPA) is emerging as a better alternative for large-scale protein quantification, the conventional TPA doesn't consider differential sequence coverage by identifying unique peptides across proteins. Here, we optimized the TPA approach by correcting protein abundance data by the sequence coverage (SC-TPA), which was applied to quantify 54 DMETs for characterization of i) differential tissue DMET abundance in the human liver, kidney, and intestine, and ii) interindividual variability of DMET proteins in individual intestinal samples (n=13). UGT2B7, MGST1, MGST2, MGST3, CES2, and MRP2 were expressed in all three tissues, whereas, as expected CYP3A4, CYP3A5, CYP2C9, CYP4F2, UGT1A1, UGT2B17, CES1, FMO5, MRP3, and P-gp were present in the liver and intestine. The top three DMET proteins in individual tissues were: CES1>CYP2E1>UGT2B7 (liver), CES2>UGT2B17>CYP3A4 (intestine), and MGST1>UGT1A6>MGST2 (kidney). CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2 showed high interindividual variability in the intestine. These data are relevant for enhancing in vitro to in vivo extrapolation (IVIVE) of drug absorption and disposition and can be used to enhance the accuracy of physiologically based pharmacokinetic (PBPK) prediction of systemic and tissue concentration of drugs. Significance Statement We quantified the abundance and compositions of drug-metabolizing enzymes and transporters (DMETs) in pooled human liver, intestine, and kidney microsomes using an optimized sequence coverage-informed total protein approach. The quantification of DMETs revealed quantitative differences in their levels in the liver, intestine, and kidney. Further, the analysis of individual intestine samples confirmed high variability in the levels of CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2. These data are applicable for the prediction of first-pass metabolism and tissue-specific drug clearance.

A Simplified Proteomics LC-MRM-MS Assay for Determination of apoE Genotypes in Plasma Samples.

Apolipoprotein E (apoE), a polymorphic plasma protein, plays a pivotal role in lipid transportation. The human apoE gene possesses three major alleles (ε2, ε3, and ε4), which differ by single amino acid (cysteine to arginine) substitutions. The ε4 allele represents the primary genetic risk factor for Alzheimer's disease (AD), whereas the ε2 allele protects against the disease. Knowledge of a patient's apoE genotype has high diagnostic value. A recent study has introduced an LC-MRM-MS-based proteomic approach for apoE isoform genotyping using stable isotope-labeled peptide internal standards (SIS). Here, our goal was to develop a simplified LC-MRM-MS assay for identifying apoE genotypes in plasma samples, eliminating the need for the use of SIS peptides. To determine the apoE genotypes, we monitored the chromatographic peak area ratios of isoform-specific peptides relative to a peptide that is common to all apoE isoforms. The assay results correlated well with the standard TaqMan allelic discrimination assay, and we observed a concordance between the two methods for all but three out of 172 samples. DNA sequencing of these three samples has confirmed that the results of the LC-MRM-MS method were correct. Thus, our simplified UPLC-MRM-MS assay is a feasible and reliable method for identifying apoE genotypes without using SIS internal standard peptides. The approach can be seamlessly incorporated into existing quantitative proteomics assays and kits, providing additional valuable apoE genotype information. The principle of using signal ratios of the protein isoform-specific peptides to the peptide common for all of the protein isoforms has the potential to be used for protein isoform determination in general.

Discovery, verification, and validation of walnut protein marker peptides using LC-MS approaches.

A key requirement of liquid chromatography-mass spectrometry (LC-MS)-based allergenic food protein analysis methods is to use protein marker peptides with good analytical performances in LC-MS analysis of commercial processed foods. In this study, we developed a multi-stage walnut protein marker peptide selection strategy involving marker peptide discovery and verification and LC-MS validation of chemically equivalent stable isotope-labeled peptides. This strategy proposed three walnut protein marker peptides, including two new marker peptides. Our LC-MS-based walnut protein analysis method using the three stable isotope-labeled peptides showed acceptable linearity (R2>0.99), matrix effects (coefficient of variation <±15%), sensitivity (limit of detection>0.3pg/μL, limit of quantification>0.8pg/μL), recovery (85.1-103.4%), accuracy, and precision (coefficient of variation<10%). In conclusion, our multi-stage marker peptide selection strategy effectively selects specific protein marker peptides for sensitive detection and absolute quantification of walnut proteins in LC-MS analysis of commercial processed foods.

Characterization and Quantification of Von Willebrand Factor Proteoforms in Von Willebrand Disease Patients and Healthy Controls: Insights from the Win-Study

Background: Von Willebrand disease (VWD) is a commonly inherited bleeding disorder which is characterized by either low (type 1) or absent (type 3) levels of Von Willebrand factor (VWF) or by the occurrence of dysfunctional VWF proteoforms (type 2) in plasma. The complexity of diagnosing and managing VWD arises from limited understanding of the diverse VWF proteoforms present in both healthy individuals as well as in VWD patients. Objective: The aim of this study was to investigate the plasma proteome and to characterize VWF proteoforms in Von Willebrand disease (VWD) patients, that were enrolled in the Willebrand in the Netherlands (WiN) 1 study, compared with healthy controls. Method: We selected type 1, type 2A, type 2M, type 2N and type 3 patients from the WiN cohort for which extensive laboratory- and genotype data were available. Plasma from 64 VWD patients and ten healthy controls was analyzed using a combination of discovery- and targeted mass spectrometry-based approaches. Results: Both discovery-based as well as targeted quantitative proteomics analysis revealed significant variation in VWF levels across the selected VWD patient group, which was in line with reported clinical laboratory data. Elevated VWF-propeptide/VWF ratios were observed in plasma samples from a range of VWD patients, with notable increases observed in VWD type 2A, B, and M, indicating accelerated clearance of these VWF proteoforms. Discovery-based proteomics revealed a strong VWF-F8 relationship in all VWD subtypes and healthy controls, except for type 2N VWD. We were able to identify peptides corresponding with common polymorphisms ( e.g. p.Thr798Ala, p.Gln852Arg, p.Thr1381Ala), for which the observed peptide distribution was in line with the minor allele frequency reported by the Broad institute. In addition, we identified peptides corresponding with 16 pathogenic VWF variants (e.g. p.Arg1306Trp, p. Phe1293Leu, p.Cys1190Arg, p.Arg1374His or p.Tyr1584Cys) of which p.Cys1190Arg wild-type and variant peptide aligned with the zygosity at DNA level (Figure X). Based on spike-in with stable isotope-labeled peptides, the variant-to-wildtype ratios varied among VWD patients with distinct pathogenic variants, ranging from 0.5-2.5 in type 2M (p.Phe1293Leu) and type 1 patients (p.Tyr1584Cys), &amp;lt;1 in type 2A patients (p.Arg1374His), to &amp;gt;8 in type 2B patients (p.Arg1306Trp), suggesting distinct stoichiometry of VWF variants among these patients (Figure 2). Conclusion: Our study highlights the feasibility of VWF proteoform identification and quantification from plasma and suggest a potential role in unravelling their contribution to the biological variations and clinical diversity observed in VWD. 1 De Wee et al., Determinants of bleeding phenotype in adult patients with moderate or severe von Willebrand disease. Thromb Haemost. 2012 Oct;108(4):683-92.

Quantitative Proteomics of Maternal Blood Plasma in Isolated Intrauterine Growth Restriction

Intrauterine growth restriction (IUGR) remains a significant concern in modern obstetrics, linked to high neonatal health problems and even death, as well as childhood disability, affecting adult quality of life. The role of maternal and fetus adaptation during adverse pregnancy is still not completely understood. This study aimed to investigate the disturbance in biological processes associated with isolated IUGR via blood plasma proteomics. The levels of 125 maternal plasma proteins were quantified by liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) with corresponding stable isotope-labeled peptide standards (SIS). Thirteen potential markers of IUGR (Gelsolin, Alpha-2-macroglobulin, Apolipoprotein A-IV, Apolipoprotein B-100, Apolipoprotein(a), Adiponectin, Complement C5, Apolipoprotein D, Alpha-1B-glycoprotein, Serum albumin, Fibronectin, Glutathione peroxidase 3, Lipopolysaccharide-binding protein) were found to be inter-connected in a protein–protein network. These proteins are involved in plasma lipoprotein assembly, remodeling, and clearance; lipid metabolism, especially cholesterol and phospholipids; hemostasis, including platelet degranulation; and immune system regulation. Additionally, 18 proteins were specific to a particular type of IUGR (early or late). Distinct patterns in the coagulation and fibrinolysis systems were observed between isolated early- and late-onset IUGR. Our findings highlight the complex interplay of immune and coagulation factors in IUGR and the differences between early- and late-onset IUGR and other placenta-related conditions like PE. Understanding these mechanisms is crucial for developing targeted interventions and improving outcomes for pregnancies affected by IUGR.

M-protein diagnostics in multiple myeloma patients using ultra-sensitive targeted mass spectrometry and an off-the-shelf calibrator.

Minimal residual disease status in multiple myeloma is an important prognostic biomarker. Recently, personalized blood-based targeted mass spectrometry (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to measure minimal residual disease. However, quantification of MS-MRD requires a unique calibrator for each patient. The use of patient-specific stable isotope labelled (SIL) peptides is relatively costly and time-consuming, thus hindering clinical implementation. Here, we introduce a simplification of MS-MRD by using anoff-the-shelf calibrator. SILuMAB-based MS-MRD was performed by spiking a monoclonal stable isotope labeled IgG, SILuMAB-K1, in the patient serum. The abundance of both M-protein-specific peptides and SILuMAB-specific peptides were monitored by mass spectrometry. The relative ratio between M-protein peptides and SILuMAB peptides allowed for M-protein quantification. We assessed linearity, sensitivity and reproducibility of SILuMAB-based MS-MRD in longitudinally collected sera from the IFM-2009 clinical trial. A linear dynamic range was achieved of over 5 logscales, allowing for M-protein quantification down to 0.001 g/L. The inter-assay CV of SILuMAB-based MS-MRD wason average 11 %. Excellent concordance between SIL-and SILuMAB-based MS-MRD was shown (R2>0.985). Additionally, signal intensity of spiked SILuMAB can be used for quality control purpose to assess system performance and incomplete SILuMAB digestion can be used as quality control for sample preparation. Compared to SIL peptides, SILuMAB-based MS-MRD improves the reproducibility, turn-around-times and cost-efficacy of MS-MRD without diminishing its sensitivity and specificity. Furthermore, SILuMAB can be used asa MS-MRD quality control tool to monitor sample preparation efficacy and assay performance.

Synthesis of S-Carbamidomethyl Cysteine and Its Use for Quantification of Cysteinyl Peptides by Targeted Proteomics.

Proteomics has played a central role in the identification of reliable disease biomarkers, which are the basis of precision medicine, a promising approach for tackling recalcitrant diseases such as cancer, that elude conventional treatments. Among proteomic methodologies, targeted proteomics employing stable isotope-labeled (SIL) internal standards is particularly suited for the clinical translation of biomarker information owing to its high throughput and accuracy in the quantitative analysis of patient-derived proteomes. Using SIL internal standards ensures the utmost level of confidence in detection and precision in targeted MS experiments. For successfully establishing assays based on targeted proteomics, it is crucial to secure broad coverage when selecting the SIL standard peptide panel. However, cysteinyl peptides have often been excluded because of cysteine's high chemical reactivity. To address this limitation, a new cysteine building block was developed by incorporating a sulfhydryl group configured with an S-carbamidomethyl group, which is commonly used in proteome sampling. This compound was found to be chemically stable and applicable to a variety of solid-phase peptide synthesis (SPPS) campaigns. Furthermore, a direct comparison of the synthesized SIL peptides and tryptic endogenous peptides demonstrated the potential utility of an SPPS flow based on the new cysteine building block for improving the success of targeted proteomic applications.

Simultaneous detection of glycinin and β-conglycinin in processed soybean products by high-performance liquid chromatography-tandem mass spectrometry with stable isotope-labeled standard peptides

Glycinin and β-conglycinin are the two main allergic proteins in soybean. Due to their complex structures and lack of protein standards, it is difficult to achieve quantitative determination of these proteins in soybeans. In this study, an HPLC-MS/MS method was developed for the simultaneous determination of five subunits of glycinin (G1, G2, G3, G4, and G5) and three subunits of β-conglycinin (α, α', and β) in processed soybean products based on 8 specific peptides and their stable isotope-labeled peptides. Here, each specific peptide was derived from one of the above 8 subunits. When soy protein was extracted and digested with trypsin, 8 specific peptides, and corresponding stable isotope-labeled peptides were analyzed by HPLC-MS/MS. The linear range for the specific peptides was between 3.2 and 1000 ng/mL (R2 > 0.9955). The recoveries of added peptides ranged from 83.4% to 117.8%, and the intra-day precisions (% CV) were below 17.4%. The limit of quantification of each subunit of glycinin and β-conglycinin in processed soybean products (in terms of protein amount) was between 15.1 and 156.1 g/g. This method was successfully applied to the analysis of 8 subunits of glycinin and β-conglycinin in 68 different processed soybean products, which provides technical support for processed product quality evaluation and monitoring soybean processing technology.

Monitoring Both Extended and Tryptic Forms of Stable Isotope-Labeled Standard Peptides Provides an Internal Quality Control of Proteolytic Digestion in Targeted Mass Spectrometry-Based Assays

Targeted mass spectrometry (MS)-based proteomic assays, such as multiplexed multiple reaction monitoring (MRM)-MS assays, enable sensitive and specific quantification of proteotypic peptides as stoichiometric surrogates for proteins. Efforts are underway to expand the use of MRM-MS assays in clinical environments, which requires a reliable strategy to monitor proteolytic digestion efficiency within individual samples. Towards this goal, extended stable isotope-labeled standard (SIS) peptides (hE), which incorporate native proteolytic cleavage sites, can be spiked into protein lysates prior to proteolytic (trypsin) digestion, and release of the tryptic SIS peptide (hT) can be monitored. However, hT measurements alone cannot monitor the extent of digestion and may be confounded by matrix effects specific to individual patient samples; therefore, they are not sufficient to monitor sample-to-sample digestion variability. We hypothesized that measuring undigested hE, along with its paired hT, would improve detection of digestion issues compared to only measuring hT. We tested the ratio of the SIS pair measurements, or hE/hT, as a quality control (QC) metric of trypsin digestion for two MRM assays: a direct-MRM (398 targets) and an immuno-MRM (126 targets requiring immunoaffinity peptide enrichment) assay, with extended SIS peptides observable for 54% (216) and 62% (78) of the targets, respectively. We evaluated the quantitative bias for each target in a series of experiments that adversely affected proteolytic digestion (e.g., variable digestion times, pH, and temperature). We identified a subset of SIS pairs (36 for the direct-MRM, 7 for the immuno-MRM assay) for which the hE/hT ratio reliably detected inefficient digestion that resulted in decreased assay sensitivity and unreliable endogenous quantification. The hE/hT ratio was more responsive to a decrease in digestion efficiency than a metric based on hT measurements alone. For clinical-grade MRM-MS assays, this study describes a ready-to-use QC panel and also provides a road map for designing custom QC panels.

Targeted Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Regulated by H3K36me3.

Trimethylation of lysine 36 on histone H3 (H3K36me3), an epigenetic mark associated with actively transcribed genes, plays an important role in multiple cellular processes, including transcription elongation, DNA methylation, DNA repair, etc. Aberrant expression and mutations of the main methyltransferase for H3K36me3, i.e., SET domain-containing 2 (SETD2), were shown to be associated with various cancers. Here, we performed targeted profiling of 154 epitranscriptomic reader, writer, and eraser (RWE) proteins using a scheduled liquid chromatography-parallel-reaction monitoring (LC-PRM) method coupled with the use of stable isotope-labeled (SIL) peptides as internal standards to investigate how H3K36me3 modulates the chromatin occupancies of epitranscriptomic RWE proteins. Our results showed consistent changes in chromatin occupancies of RWE proteins upon losses of H3K36me3 and H4K16ac and a role of H3K36me3 in recruiting METTL3 to chromatin following induction of DNA double-strand breaks. In addition, protein-protein interaction network and Kaplan-Meier survival analyses revealed the importance of METTL14 and TRMT11 in kidney cancer. Taken together, our work unveiled cross-talks between histone epigenetic marks (i.e., H3K36me3 and H4K16ac) and epitranscriptomic RWE proteins and uncovered the potential roles of these RWE proteins in H3K36me3-mediated biological processes.

Sample Preparation Methods for Targeted Single-Cell Proteomics.

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540-660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390-540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5-25 RBCs obtained from N = 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.

Targeted Proteomic Profiling Revealed Roles of Small GTPases during Osteogenic Differentiation.

The small GTPase superfamily of proteins are crucial for numerous cellular processes, including early development. The roles of these proteins in osteogenic differentiation, however, remained poorly explored. In this study, we employed a high-throughput targeted proteomic method, relying on scheduled liquid chromatography-multiple-reaction monitoring (LC-MRM) coupled with synthetic stable isotope-labeled peptides, to interrogate systematically the temporal responses of the entire small GTPase proteome during the course of osteogenic differentiation of H9 human embryonic stem cells. Our results demonstrated that the method offers high quantification accuracy, reproducibility, and throughput. In addition, the quantification results revealed altered expression of a large number of small GTPases accompanied with osteogenic differentiation, especially those involved with autophagy. We also documented a previously unrecognized role of KRAS in osteogenesis, where it regulates the accumulation of extracellular matrix for mineralization through attenuating the activity of secreted matrix metalloproteinase 9 (MMP9). Together, this study represents a novel application of a state-of-the-art analytical method, i.e., targeted quantitative proteomics, for revealing the progressive reprogramming of the small GTPase proteome during osteogenic differentiation of human embryonic stem cells, and our results revealed KRAS as a new regulator for osteogenesis.

Proteomic identification and quantification of Clostridium perfringens enterotoxin using a stable isotope-labelled peptide via liquid chromatography-tandem mass spectrometry.

Detection of Clostridium perfringens enterotoxin (CPE) in human stool is critical evidence of food poisoning. However, processing patient-derived samples is difficult and very few methods exist to confirm the presence of CPE. In this study, a technique was developed using proteomic analysis to identify and quantify CPE in artificial gut fluid as an alternative. The standard CPE was spiked into artificial gut fluids, and effective methods were developed by employing both a stable isotope-labelled internal standard peptide and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteotypic peptide EILDLAAATER formed by tryptic digestion was selected for quantitation of CPE. The peptide was identified using product ion spectra. Although the nontoxic peptides originating from CPE showed very low detectability in extraction and tryptic digestion, they could be detected with sufficient sensitivity using the method we developed. Based on a spiked recovery test at two concentrations (50 and 200µg/kg), the recovery values were 85 and 78%, respectively. The relative standard deviations of repeatability and within-laboratory reproducibility were less than 8 and 11%, respectively. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification (LOQ) was estimated to be 50µg/kg. The combination of the product ion spectra and relative ion ratio supported CPE identification at the LOQ level. To the best of our knowledge, this is the first report of proteomic analysis of CPE using LC-MS/MS. The method would greatly help in assessing CPE reliably.

AlacatDesigner─Computational Design of Peptide Concatamers for Protein Quantitation.

Protein quantitation via mass spectrometry relies on peptide proxies for the parent protein from which abundances are estimated. Owing to the variability in signal from individual peptides, accurate absolute quantitation usually relies on the addition of an external standard. Typically, this involves stable isotope-labeled peptides, delivered singly or as a concatenated recombinant protein. Consequently, the selection of the most appropriate surrogate peptides and the attendant design in recombinant proteins termed QconCATs are challenges for proteome science. QconCATs can now be built in a "a-la-carte" assembly method using synthetic biology: ALACATs. To assist their design, we present "AlacatDesigner", a tool that supports the peptide selection for recombinant protein standards based on the user's target protein. The user-customizable tool considers existing databases, occurrence in the literature, potential post-translational modifications, predicted miscleavage, predicted divergence of the peptide and protein quantifications, and ionization potential within the mass spectrometer. We show that peptide selections are enriched for good proteotypic and quantotypic candidates compared to empirical data. The software is freely available to use either via a web interface AlacatDesigner, downloaded as a Desktop application or imported as a Python package for the command line interface or in scripts.

Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-μm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine–based immunohistochemical staining. We analyzed serial unstained and stained sections from non–small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted β-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.

An Old Pathway Learns Two New Tricks: Role of Complement in Postoperative Cognitive Dysfunction, and in Synaptic Damage in APOE4 carriers

AbstractBackgroundGenetic studies in both mice and humans suggest a role for complement in Alzheimer's disease, but exactly how complement contributes to preclinical AD progression is unclear. It is also unclear to what extent complement may play a role in AD risk within APOE4 carriers, or in the pathophysiology of perioperative neurocognitive disorders in older adults.MethodWe developed a hybrid proteomics platform, which uses both LC‐MS/MS for unbiased discovery across the entire human proteome, as well as stable isotope labelled (SIL) peptides for complement proteins to precisely quantify complement protein‐derived peptides (as would normally be possible only in a targeted proteomics assay). This platform was then used to study proteins and pathways altered in the CSF of APOE4 carriers vs non‐carriers, in which the vast majority of both groups were amyloid negative. We also used this platform to study pre‐operative samples from patients who did and those who did not develop postoperative delirium. Generally Applicable Gene Set Enrichment (GAGE) analysis was used to study alterations within specific protein pathways in both APOE4 carriers, and in patients with postoperative cognitive dysfunction.ResultIn our unbiased data, we detected &gt;9,000 different peptides from 884 different proteins in over 100 CSF samples from older adults with normal cognition, including over 200 different complement protein‐derived peptides. In the complement proteins for which we had SIL peptides, we found an average reduction of ∼8.4% ± 0.5% in the levels of more than 50 peptides from each protein in the classical, alternative and mannose binding lectin complement cascades in APOE4 homozygotes versus APOE4 non‐carriers; the odds of this happening by chance would be 1 out of 2 x10^15. We also found reductions in complement proteins (and a significant change in the complement pathway as a whole) at multiple time points before and after surgery in patients who developed postoperative cognitive dysfunction.ConclusionThese results suggest that CNS complement pathway activation may play a role in both postoperative cognitive dysfunction, and in APOE4‐induced brain damage even in the absence of amyloid pathology.

APOE4 copy number‐dependent Complement Pathway Dysregulation

AbstractBackgroundComplement has been implicated in the pathophysiology of AD based on human genetic studies, human post‐mortem pathology, and mouse genetic studies. However, the relationship between complement activation and other AD risk factors (such as the APOE4 allele) remains unclear.MethodWe used pathway analysis of the ADNI targeted proteomics dataset to identify protein clusters highly correlated with each other, including a complement pathway cluster. We then developed a hybrid proteomics platform, which allows for both discovery proteomics (via LC‐MS/MS) and precise quantitation of complement protein‐derived peptides via Stable Isotope Labelled (SIL) internal control peptides for the complement proteins. We then tested this hybrid proteomics platform on over 130 CSF samples from cognitively normal older adults (ages 60‐80), together with assays for CSF amyloid beta, tau and p‐tau‐181p. Spearman correlation tests were used to evaluate correlations between protein pathway expression and demographic variables, such as APOE4 allele number.ResultPathway analysis of the ADNI targeted proteomics dataset identified a cluster of complement proteins whose levels were highly correlated with each other in the CSF; this cluster included peptides from C1QB, C2, C3, C4A, C5, C6, C8B and CRP and several additional proteins. Across ∼300 individuals (including cognitively normal controls, MCI and AD patients), expression of this complement protein cluster was inversely correlated with APOE4 allele count and gender (with lower expression in females), and a positive correlation with age (P&lt;0.05 for each). To further study complement protein levels in human CSF across the AD spectrum, we developed a hybrid proteomics platform for precise quantitation of complement proteins and for unbiased discovery proteomics. In 130 CSF samples from older adults, we were able to detect ∼9,000 different peptides from 884 different proteins, and to precisely quantitate all proteins in the classical, alternative and mannose binding lectin complement pathways using the SIL peptide internal controls. Here we report data on APOE4 copy number dependent changes in quantified CSF complement protein levels, and on APOE4 dependent changes across the human CSF proteome.ConclusionAPOE4 is associated with a reduction in CSF complement protein levels, which may reflect increased complement pathway activation and ensuing degradation of these proteins.

Development of simple and rapid method for Emicizumab quantification by LC-MS/MS in human plasma

Emicizumab is a new therapeutic monoclonal antibody indicated for prophylaxis in severe haemophilia A patients. Pharmacokinetic variability has been reported in clinical studies, thus dose optimisation based on quantification of plasma drug concentration could be considered to reduce this variability. Therefore, a reliable and accurate quantification of emicizumab is required. In this study, we developed a method for absolute quantification of emicizumab using liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). Sample preparation was based on organic solvent precipitation of proteins followed by trypsin digestion. A signature peptide of emicizumab was used for quantification by LC-MS/MS. A stable isotope-labelled peptide was used as an internal standard. Finally, 6 samples from patients treated with emicizumab were quantified by LC-MS/MS and compared with those obtained with the modified one-stage activated partial prothrombin time technique (aPTT) based FVIII activity. The LC-MS/MS method was validated according to FDA recommendations. Good linearity of the calibration curves was observed over the range 5–150 µg/mL. The cross-validation showed an acceptable correlation of the developed LC-MS/MS method with the modified aPTT-based FVIII activity assay, and the Bland-Altman analysis did not show any significant bias.

Minimal Residual Disease Monitoring By Targeted Mass Spectrometry Allows Early Relapse Detection in Multiple Myeloma

Background Multiple myeloma (MM) is a clonal plasma cell disorder found in the bone marrow that produces a monoclonal immunoglobulin (M-protein). With blood based electrophoretic assays such as serum protein electrophoresis (SPEP) the M-protein is monitored, but with limited sensitivity. Minimal residual disease (MRD) negativity during remission correlates with prolonged progression free survival. Lack of sensitivity prevents MRD detection by conventional blood based assays. Bone marrow based assays such as next-generation sequencing (NGS) are highly sensitive in measuring MRD. However, bone marrow biopsies introduce a risk of non-representative sampling and are invasive, which limits repeated testing. Frequent MM monitoring during remission could provide actionable information on disease activity and treatment response. Earlier detection of disease progression could lead to early intervention and, potentially, patient survival benefits. Aim The aim of this study was to perform M-protein monitoring on blood samples of MM patients with sensitive targeted mass spectrometry (MS-MRD), to show the capabilities of this MRD-approach. Methods Forty-one MM patients were selected from the IFM 2009 study (ClinicalTrials.gov number: NCT01191060) with 13-31 serum samples per patient collected longitudinally over 29-60 months follow-up. RNA sequences, NGS-MRD, and SPEP data were available to us. All patients were treated with lenalidomide, bortezomib, and dexamethasone, but only treatment arm B received autologous stem-cell transplantation (ASCT). For each patient, 2 unique tryptic peptides from the M-protein were selected, 1 peptide was selected for patients with only light chain M-protein. For early increases in disease activity, absolute M-protein quantification (g/dL) was performed based on tryptic peptides and stable isotope-labeled peptide as an internal standard. Results Out of a total of 929 measured samples, M-protein was detected in 879 samples by MS-MRD and only in 195 samples by SPEP. Furthermore, MS-MRD showed a 78% concordance with NGS-MRD with 14 samples being MS-MRD positive but NGS-MRD negative. Sensitivity can vary per patient and was estimated between 0.03-0.57 mg/dL in this cohort. Based on the M-protein profiles obtained with MS-MRD, during the maintenance treatment, increase of disease activity was observed in a subset of patients from arm A whereas all patients from arm B, who received ASCT, showed a persistent M-protein decrease. An increase of MM disease activity was observed earlier with MS-MRD in 13 out of 14 patients known to progress during the serum collection period, with a median of 455 days gained compared to the original trial data (p<0.0001). Figure 1 shows MS-MRD data showing disease activity 16 months prior to the known progression. Conclusion A non-invasive and sensitive MS-MRD assay for frequent MM monitoring was implemented on clinical trial samples. MS-MRD showed that patients who received ASCT had a more durable response with decreasing M-protein levels. Moreover, in 93% of the cases an increase of disease activity was detected earlier than with conventional blood tests by more than a year. Future perspective The dynamic information on disease activity that is provided by the MS-MRD can provide patients with feedback on their disease status after each checkup, and can provide clinicians options for evidence based treatment escalation or de-escalation. While the overall patient benefit of such personalized treatment still needs to be assessed in clinical trials, we expect that this can improve patient quality of life and allow for more efficient use of pharmacotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeted Proteomic Analysis of Small GTPases in Radioresistant Breast Cancer Cells.

Radiation therapy benefits more than 50% of all cancer patients and cures 40% of them, where ionizing radiation (IR) deposits energy to cells and tissues, thereby eliciting DNA damage and resulting in cell death. Small GTPases are a superfamily of proteins that play critical roles in cell signaling. Several small GTPases, including RAC1, RHOB, and RALA, were previously shown to modulate radioresistance in cancer cells. However, there is no systematic proteomic study on small GTPases that regulate radioresistance in cancer cells. Herein, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled (SIL) peptides, to identify differentially expressed small GTPase proteins in two pairs of breast cancer cell lines, MDA-MB-231 and MCF7, and their corresponding radioresistant cell lines. We identified 7 commonly altered small GTPase proteins with over 1.5-fold changes in the two pairs of cell lines. We also discovered ARFRP1 as a novel regulator of radioresistance, where its downregulation promotes radioresistance in breast cancer cells. Together, this represents the first comprehensive investigation about the differential expression of the small GTPase proteome associated with the development of radioresistance in breast cancer cells. Our work also uncovered ARFRP1 as a new target for enhancing radiation sensitivity in breast cancer.