Background: Becker muscular dystrophy (BMD) is a rare and heterogeneous form of dystrophinopathy caused by expression of altered dystrophin proteins, as a consequence of in-frame genetic mutations. The majority of the BMD biomarker studies employ targeted approaches and focus on translating findings from Duchenne Muscular Dystrophy (DMD), a more severe disease form with clinical similarities but caused by out-of-frame mutations in the dystrophin gene. Importantly, DMD therapies assume that disease progression can be slowed by promoting the expression of truncated dystrophin comparable to what occurs in BMD patients. In this study, we explore similarities and differences in protein trajectories over time between BMD and DMD serum, and explore proteins related to motor function performance.

Methods: Serum samples collected from 34 BMD patients, in a prospective longitudinal 3-year study, and 19 DMD patients, were analyzed by using Data Independent Acquisition Tandem Mass Spectrometry (DIA-MS). Subsequent normalization, linear mixed effects model was employed to identify proteins associated with physical tests and dystrophin expression in skeletal muscle. Analysis was also performed to explore the discrepancy between DMD and BMD biomarker abundance trajectories over time.

Results: Linear mixed effects models identified 20 proteins with altered longitudinal signatures between DMD and BMD, including creatine kinase M-type (CKM) pyruvate kinase (PKM), fibrinogen gamma chain (FGG), lactate dehydrogenase B (LDHB) and alpha-2-macroglobulin (A2M). Furthermore, several proteins related to innate immune response were associated with motor function in BMD patients. In particular, A2M displayed an altered time-dependent decline in relation to dystrophin expression in the tibialis anterior muscle.

Conclusions: Our study revealed differences in the serum proteome between BMD and DMD, which comprises proteins involved in the immune response, extracellular matrix organization and hemostasis but not muscle leakage proteins significantly associated with disease progression in DMD. If further evaluated and validated, these biomarker candidates may offer means to monitor disease progression in BMD patients. A2M is of particular interest due to its association with dystrophin expression in BMD muscle and higher abundance in DMD patients in comparison to BMD. If validated, A2M could be used as a pharmacodynamic biomarker in therapeutic clinical trials aiming to restore dystrophin expression.

The creatine-phosphocreatine cycle serves as a crucial temporary energy buffering system in the brain, regulated by brain creatine kinase (CKB), in maintaining Adenosine triphosphate (ATP) levels. Alzheimer's disease (AD) has been linked to increased CKB oxidation and loss of its regulatory function, although specific pathological processes and affected cell types remain unclear. In our study, cerebral cortex samples from individuals with AD, dementia with Lewy bodies (DLB), and age-matched controls were analyzed using antibody-based methods to quantify CKB levels and assess alterations associated with disease processes. Two independently validated antibodies exclusively labeled astrocytes in the human cerebral cortex. Combining immunofluorescence (IF) and mass spectrometry (MS), we explored CKB availability in AD and DLB cases. IF and Western blot analysis demonstrated a loss of CKB immunoreactivity correlated with increased plaque load, severity of tau pathology, and Lewy body pathology. However, transcriptomics data and targeted MS demonstrated unaltered total CKB levels, suggesting posttranslational modifications (PTMs) affecting antibody binding. This aligns with altered efficiency at proteolytic cleavage sites indicated in the targeted MS experiment. These findings highlight that the proper function of astrocytes, understudied in the brain compared with neurons, is highly affected by PTMs. Reduction in ATP levels within astrocytes can disrupt ATP-dependent processes, such as the glutamate-glutamine cycle. As CKB and the creatine-phosphocreatine cycle are important in securing constant ATP availability, PTMs in CKB, and astrocyte dysfunction may disturb homeostasis, driving excitotoxicity in the AD brain. CKB and its activity could be promising biomarkers for monitoring early-stage energy deficits in AD.

Mass spectrometry based on data-independent acquisition (DIA) has developed into a powerful quantitative tool with a variety of implications, including precision medicine. Combined with stable isotope recombinant protein standards, this strategy provides confident protein identification and precise quantification on an absolute scale. Here, we describe a comprehensive targeted proteomics approach to profile a pan-cancer cohort consisting of 1800 blood plasma samples representing 15 different cancer types. We successfully performed an absolute quantification of 253 proteins in multiplex. The assay had low intra-assay variability with a coefficient of variation below 20% (CV = 17.2%) for a total of 1013 peptides quantified across almost two thousand injections. This study identified a potential biomarker panel of seven protein targets for the diagnosis of multiple myeloma patients using differential expression analysis and machine learning. The combination of markers, including the complement C1 complex, JCHAIN, and CD5L, resulted in a prediction model with an AUC of 0.96 for the identification of multiple myeloma patients across various cancer patients. All these proteins are known to interact with immunoglobulins.

Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large-scale biomedical studies. Proteases are a central component of every bottom-up proteomics experiment, digesting proteins into peptides. Trypsin has been the most widely applied protease in proteomics due to its characteristics. With ever-larger cohort sizes and possible future clinical application of mass spectrometry-based proteomics, the technical impact of trypsin becomes increasingly relevant. To assess possible biases introduced by trypsin digestion, we evaluated the impact of eight commercially available trypsins in a variety of bottom-up proteomics experiments and across a range of protease concentrations and storage times. To investigate the universal impact of these technical attributes, we included bulk HeLa cell lysate, human plasma, and single HEK293 cells, which were analyzed over a range of selected reaction monitoring (SRM), data-independent acquisition (DIA), and data-dependent acquisition (DDA) instrument methods on three LC-MS instruments. The quantification methods employed encompassed both label-free approaches and absolute quantification utilizing spike-in heavy-labeled recombinant protein fragment standards. Based on this extensive data set, we report variations between commercial trypsins, their source, and their concentration. Furthermore, we provide suggestions on the handling of trypsin in large-scale studies.

Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker.

Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering for biotechnology. Here we apply limited proteolysis-small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions shows that some metabolites interact in a species-specific manner. We estimate that approximately 35% of interacting metabolites affect enzyme activity in vitro, and the effect is often minor. Using LiP-SMap data as a guide, we find that the Calvin cycle intermediate glyceraldehyde-3-phosphate enhances activity of fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) from Synechocystis sp. PCC 6803 and Cupriavidus necator in reducing conditions, suggesting a convergent feed-forward activation of the cycle. In oxidizing conditions, glyceraldehyde-3-phosphate inhibits Synechocystis F/SBPase by promoting enzyme aggregation. In contrast, the glycolytic intermediate glucose-6-phosphate activates F/SBPase from Cupriavidus necator but not F/SBPase from Synechocystis. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated.

Lipoprotein(a), also known as Lp(a), is an LDL-like particle composed of apolipoprotein(a) (apo(a)) bound covalently to apolipoprotein B100. Plasma concentrations of Lp(a) are highly heritable and vary widely between individuals. Elevated plasma concentration of Lp(a) is considered as an independent, causal risk factor of cardiovascular disease (CVD). Targeted mass spectrometry (LC-SRM/MS) combined with stable isotope-labeled recombinant proteins provides robust and precise quantification of proteins in the blood, making LC-SRM/ MS assays appealing for monitoring plasma proteins for clinical implications. This study presents a novel quantitative approach, based on proteotypic peptides, to determine the absolute concentration of apo(a) from two microliters of plasma and qualified according to guideline requirements for targeted proteomics assays. After optimization, assay parameters such as linearity, lower limits of quantification (LLOQ), intra-assay variability (CV: 4.7%) and inter-assay repeatability (CV: 7.8%) were determined and the LC-SRM/MS results were benchmarked against a commercially available immunoassay. In summary, the measurements of an apo(a) single copy specific peptide and a kringle 4 specific peptide allow for the determination of molar concentration and relative size of apo(a) in individuals.

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. 

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.

Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering to improve industrial phenotypes. Recently developed chemoproteomics workflows allow for genome-wide detection of metabolite-protein interactions that may regulate pathway activity. We applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions showed that some metabolites interacted in a species-specific manner, such as interactions of glucose-6-phosphate in Cupriavidus necator and of glyoxylate in Synechocystis sp PCC 6803. These are interpreted in light of the different central carbon conversion pathways present. Metabolites interacting with the Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activity in vitro. The Calvin cycle intermediate glyceraldehyde-3-phosphate activated both Synechocystis and Cupriavidus F/SBPase, which suggests a feed-forward activation of the cycle in both photoautotrophs and chemolithoautotrophs. In contrast to the stimulating effect in reduced conditions, glyceraldehyde-3-phosphate inactivated the Synechocystis F/SBPase in oxidized conditions by accelerating protein aggregation. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated and may act in addition to redox regulation.