Targeted Quantification of Proteoforms in Complex Samples by Proteoform Reaction Monitoring.

Existing mass spectrometric assays used for sensitive and specific measurements of target proteins across multiple samples, such as selected/multiple reaction monitoring (SRM/MRM) or parallel reaction monitoring (PRM), are peptide-based methods for bottom-up proteomics. Here, we describe an approach based on the principle of PRM for the measurement of intact proteoforms by targeted top-down proteomics, termed proteoform reaction monitoring (PfRM). We explore the ability of our method to circumvent traditional limitations of top-down proteomics, such as sensitivity and reproducibility. We also introduce a new software program, Proteoform Finder (part of ProSight Native), specifically designed for the easy analysis of PfRM data. PfRM was initially benchmarked by quantifying three standard proteins. The linearity of the assay was shown over almost 3 orders of magnitude in the femtomole range, with limits of detection and quantification in the low femtomolar range. We later applied our multiplexed PfRM assay to complex samples to quantify biomarker candidates in peripheral blood mononuclear cells (PBMCs) from liver-transplanted patients, suggesting their possible translational applications. These results demonstrate that PfRM has the potential to contribute to the accurate quantification of protein biomarkers for diagnostic purposes and to improve our understanding of disease etiology at the proteoform level.

A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a kcat /Km value of 6.1×106  L mol-1 s-1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.

Synthesis of peptides containing 2-oxohistidine residues and their characterization by liquid chromatography-tandem mass spectrometry.

Protein oxidation by reactive oxygen species has been associated with aging and neurodegenerative disorders, and histidine is one of the major oxidation targets due to its metal-chelating property and susceptibility to metal-catalyzed oxidation. 2-Oxohistidine, the major product of histidine oxidation, has been recently identified as a stable marker of oxidative damage in biological systems, but its biophysical and biochemical properties are understudied, partly because of difficulties in its chemical synthesis. We developed an efficient method to generate a 2-oxohistidine side chain using metal-catalyzed oxidation, applicable to both monomers and peptides. By optimizing reagent ratios and pH buffering in Cu(2+) /ascorbate/O2 reaction system, we improved the yield more than tenfold compared to reported conditions, which allowed us to obtain homogeneously modified 2-oxohisidine peptides for further studies. Analysis of 2-oxohistidine-containing model peptides by liquid chromatography-tandem mass spectrometry demonstrated increased retention time in reverse-phase chromatography and general stability of 2-oxohistidine under electrospray ionization and collision-induced dissociation. Thus, large-scale analysis of 2-oxohistidine-modified proteome should be feasible using shotgun protein mass spectrometry, and we were able to observe such peptides in proteomics datasets. The feasibility of acquiring purified peptide probes and peptide antigens containing 2-oxohistidine will help advance the study of this non-enzymatic posttranslational modification.

Deep Profiling of Plasma Proteoforms with Engineered Nanoparticles for Top-down Proteomics.

The dynamic range challenge for detection of proteins and their proteoforms in human plasma has been well documented. Here, we use the nanoparticle protein corona approach to enrich low-abundant proteins selectively and reproducibly from human plasma and use top-down proteomics to quantify differential enrichment for the 2841 detected proteoforms from 114 proteins. Furthermore, nanoparticle enrichment allowed top-down detection of proteoforms between ∼1 µg/mL and ∼10 pg/mL in absolute abundance, providing up to 10 5 -fold increase in proteome depth over neat plasma in which only proteoforms from abundant proteins (>1 µg/mL) were detected. The ability to monitor medium and some low abundant proteoforms through reproducible enrichment significantly extends the applicability of proteoform research by adding depth beyond albumin, immunoglobins and apolipoproteins to uncover many involved in immunity and cell signaling. As proteoforms carry unique information content relative to peptides, this report opens the door to deeper proteoform sequencing in clinical proteomics of disease or aging cohorts.

Top-Down Proteomics Identifies Plasma Proteoform Signatures of Liver Cirrhosis Progression.

Cirrhosis, advanced liver disease, affects 2-5 million Americans. While most patients have compensated cirrhosis and may be fairly asymptomatic, many decompensate and experience life-threatening complications such as gastrointestinal bleeding, confusion (hepatic encephalopathy), and ascites, reducing life expectancy from 12 to less than 2 years. Among patients with compensated cirrhosis, identifying patients at high risk of decompensation is critical to optimize care and reduce morbidity and mortality. Therefore, it is important to preferentially direct them towards specialty care which cannot be provided to all patients with cirrhosis. We used discovery Top-down Proteomics (TDP) to identify differentially expressed proteoforms (DEPs) in the plasma of patients with progressive stages of liver cirrhosis with the ultimate goal to identify candidate biomarkers of disease progression. In this pilot study, we identified 209 DEPs across three stages of cirrhosis (compensated, compensated with portal hypertension, and decompensated), of which 115 derived from proteins enriched in the liver at a transcriptional level and discriminated the three stages of cirrhosis. Enrichment analyses demonstrated DEPs are involved in several metabolic and immunological processes known to be impacted by cirrhosis progression. We have preliminarily defined the plasma proteoform signatures of cirrhosis patients, setting the stage for ongoing discovery and validation of biomarkers for early diagnosis, risk stratification, and disease monitoring.

Mass spectrometry-based proteomics for advancing solid organ transplantation research.

Scarcity of high-quality organs, suboptimal organ quality assessment, unsatisfactory pre-implantation procedures, and poor long-term organ and patient survival are the main challenges currently faced by the solid organ transplant (SOT) field. New biomarkers for assessing graft quality pre-implantation, detecting, and predicting graft injury, rejection, dysfunction, and survival are critical to provide clinicians with invaluable prediction tools and guidance for personalized patients' treatment. Additionally, new therapeutic targets are also needed to reduce injury and rejection and improve transplant outcomes. Proteins, which underlie phenotypes, are ideal candidate biomarkers of health and disease statuses and therapeutic targets. A protein can exist in different molecular forms, called proteoforms. As the function of a protein depends on its exact composition, proteoforms can offer a more accurate basis for connection to complex phenotypes than protein from which they derive. Mass spectrometry-based proteomics has been largely used in SOT research for identification of candidate biomarkers and therapeutic intervention targets by so-called "bottom-up" proteomics (BUP). However, such BUP approaches analyze small peptides in lieu of intact proteins and provide incomplete information on the exact molecular composition of the proteins of interest. In contrast, "Top-down" proteomics (TDP), which analyze intact proteins retaining proteoform-level information, have been only recently adopted in transplantation studies and already led to the identification of promising proteoforms as biomarkers for organ rejection and dysfunction. We anticipate that the use of top-down strategies in combination with new technological advancements in single-cell and spatial proteomics could drive future breakthroughs in biomarker and therapeutic target discovery in SOT.

Tyrosine phosphatase activity is restricted by basic charge substituting mutation of substrates.

Phosphorylation controls important cellular signals and its dysregulation leads to disease. While most phospho-regulation studies are focused on kinases, phosphatases are comparatively overlooked. Combining peptide arrays with SAMDI mass spectrometry, we show that tyrosine phosphatase activity is restricted by basic amino acids adjacent to phosphotyrosines. We validate this model using two ß-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K). These mutants introduce a basic residue next to Y654, an established phosphorylation site where modification shifts ß-catenin from cell-cell adhesions and towards its essential nuclear role as Wnt-signaling effector. We show that T653-basic mutant ß-catenins are less efficiently dephosphorylated by phosphatases, leading to sustained Y654 phosphorylation and elevated Wnt signals, similar to those observed for Y654E phospho-mimic mutant mice. This model rationalizes how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6000+ potentially relevant cancer mutations.

Profiling Protein Tyrosine Phosphatase Specificity with Self-Assembled Monolayers for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Peptide Arrays.

The opposing activities of phosphatases and kinases determine the phosphorylation status of proteins, yet kinases have received disproportionate attention in studies of cellular processes, with the roles of phosphatases remaining less understood. This Research Article describes the use of phosphotyrosine-containing peptide arrays together with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to directly profile phosphatase substrate selectivities. Twenty-two protein tyrosine phosphatases were characterized with the arrays to give a profile of their specificities. An analysis of the data revealed that certain residues in the substrates had a conserved effect on activity for all enzymes tested, including the general rule that inclusion of a basic lysine or arginine residue on either side of the phosphotyrosine decreased activity. This insight also provides a new perspective on the role of a R1152Q mutant in the insulin receptor, which is known to exhibit a lower phosphorylation level and which this work suggests may be due to an increased activity toward phosphatase enzymes. The use of self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to provide a rapid and quantitative assay of phosphatase enzymes will be important to gaining a more complete understanding of the biochemistry and biology of this important enzyme class.

Combining SAMDI Mass Spectrometry and Peptide Arrays to Profile Phosphatase Activities.

Phosphatases, the enzymes responsible for dephosphorylating proteins, play critical roles in many cellular processes. While their importance is widely recognized, phosphatase activity and regulation remain poorly understood. Currently, there are few assays available that are capable of directly measuring phosphatase activity and specificity. We have previously introduced SAMDI (self-assembled monolayers on gold for matrix-assisted laser desorption/ionization) mass spectrometry as a technique to profile the substrate specificities of enzymes. SAMDI mass spectrometry assays are well suited to examine phosphatase activities and offer many advantages over current methods. This technique uses monolayers that terminate with a peptide or molecular enzyme substrate and allows for enzyme reactions to be performed on a surface that can easily be rinsed and analyzed by mass spectrometry without the need for analyte labeling. In this chapter, we describe the process of combining SAMDI mass spectrometry with peptide arrays to study the substrate specificities of two protein tyrosine phosphatases.