Promises and Challenges of populational Proteomics in Health and Disease.

Advances in proteomic assay technologies have significantly increased coverage and throughput, enabling recent increases in the number of large-scale population-based proteomic studies of human plasma and serum. Improvements in multiplexed protein assays have facilitated the quantification of thousands of proteins over a large dynamic range, a key requirement for detecting the lowest-ranging, and potentially the most disease-relevant, blood-circulating proteins. In this perspective, we examine how populational proteomic datasets in conjunction with other concurrent omic measures can be leveraged to better understand the genomic and non-genomic correlates of the soluble proteome, constructing biomarker panels for disease prediction, among others. Mass spectrometry workflows are discussed as they are becoming increasingly competitive with affinity-based array platforms in terms of speed, cost, and proteome coverage due to advances in both instrumentation and workflows. Despite much success, there remain considerable challenges such as orthogonal validation and absolute quantification. We also highlight emergent challenges associated with study design, analytical considerations, and data integration as population-scale studies are run in batches and may involve longitudinal samples collated over many years. Lastly, we take a look at the future of what the nascent next-generation proteomic technologies might provide to the analysis of large sets of blood samples, as well as the difficulties in designing large-scale studies that will likely require participation from multiple and complex funding sources and where data sharing, study designs, and financing must be solved.

Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 µg of HeLa protein. CSC also revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs.

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously "undruggable" proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12-186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target "degradability."

Identification of Proteolysis Products in Protein Therapeutics through TMPP N-Terminal Tagging and Electron Transfer Dissociation Product Triggered Collisional Induced Dissociation Fragmentation.

Thorough characterization of protein therapeutics is often challenging due to the heterogeneity arising from primary sequence variants, post-translational modifications, proteolytic clipping, or incomplete processing of the signal peptide. Modern mass spectrometry (MS) techniques are now routinely used to characterize such heterogeneous protein populations. Here, we present an LC-MS/MS method using (N-succinimidyloxycarbonylmethyl)-tris (2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to label any free N-terminal α-amines to rapidly and selectively identify proteolytic clipping events. Electron transfer dissociation (ETD) fragmentation of these chemically tagged peptides generates two unique TMPP product ions, TMPP+ and TMPP-Ac-NH2/c0. The presence of these signature ions following ETD is used to trigger subsequent collisional induced dissociation (CID) fragmentation of the precursor ion. This results in a small subset of CID tandem MS spectra that are used in a customized database search. Using a purified fusion monoclonal antibody (mAb) as an example, we demonstrate how TMPP labeling followed by ETD product ion triggered CID fragmentation is used to accurately identify two undesired clipping sites.

Proteome and Secretome Dynamics of Human Retinal Pigment Epithelium in Response to Reactive Oxygen Species.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries, and is characterized by slow retinal degeneration linked to chronic reactive oxygen species (ROS) in the retinal pigmented epithelium (RPE). The molecular mechanisms leading to RPE dysfunction in response to ROS are unclear. Here, human stem cell-derived RPE samples were stressed with ROS for 1 or 3 weeks, and both intracellular and secreted proteomes were quantified by mass spectrometry. ROS increased glycolytic proteins but decreased mitochondrial complex I subunits, as well as membrane proteins required for endocytosis. RPE secreted over 1,000 proteins, many of which changed significantly due to ROS. Notably, secreted APOE is decreased 4-fold, and urotensin-II, the strongest known vasoconstrictor, doubled. Furthermore, secreted TGF-beta is increased, and its cognate signaler BMP1 decreased in the secretome. Together, our results paint a detailed molecular picture of the retinal stress response in space and time.

Dietary Sugars Alter Hepatic Fatty Acid Oxidation via Transcriptional and Post-translational Modifications of Mitochondrial Proteins.

Dietary sugars, fructose and glucose, promote hepatic de novo lipogenesis and modify the effects of a high-fat diet (HFD) on the development of insulin resistance. Here, we show that fructose and glucose supplementation of an HFD exert divergent effects on hepatic mitochondrial function and fatty acid oxidation. This is mediated via three different nodes of regulation, including differential effects on malonyl-CoA levels, effects on mitochondrial size/protein abundance, and acetylation of mitochondrial proteins. HFD- and HFD plus fructose-fed mice have decreased CTP1a activity, the rate-limiting enzyme of fatty acid oxidation, whereas knockdown of fructose metabolism increases CPT1a and its acylcarnitine products. Furthermore, fructose-supplemented HFD leads to increased acetylation of ACADL and CPT1a, which is associated with decreased fat metabolism. In summary, dietary fructose, but not glucose, supplementation of HFD impairs mitochondrial size, function, and protein acetylation, resulting in decreased fatty acid oxidation and development of metabolic dysregulation.

SILAC Analysis Reveals Increased Secretion of Hemostasis-Related Factors by Senescent Cells.

Cellular senescence irreversibly arrests cell proliferation, accompanied by a multi-component senescence-associated secretory phenotype (SASP) that participates in several age-related diseases. Using stable isotope labeling with amino acids (SILACs) and cultured cells, we identify 343 SASP proteins that senescent human fibroblasts secrete at 2-fold or higher levels compared with quiescent cell counterparts. Bioinformatic analysis reveals that 44 of these proteins participate in hemostasis, a process not previously linked with cellular senescence. We validated the expression of some of these SASP factors in cultured cells and in vivo. Mice treated with the chemotherapeutic agent doxorubicin, which induces widespread cellular senescence in vivo, show increased blood clotting. Conversely, selective removal of senescent cells using transgenic p16-3MR mice showed that clearing senescent cells attenuates the increased clotting caused by doxorubicin. Our study provides an in-depth, unbiased analysis of the SASP and unveils a function for cellular senescence in hemostasis.

JNK modifies neuronal metabolism to promote proteostasis and longevity.

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age-related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long-lived animals with elevated Jun-N-terminal Kinase signaling. We find that these animals exhibit a delayed age-related decline in protein turnover rates, as well as decreased steady-state neuronal glucose-6-phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose-6-phosphate dehydrogenase (G6PD). Over-expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long-lived mutants.

Temporal dynamics of liver mitochondrial protein acetylation and succinylation and metabolites due to high fat diet and/or excess glucose or fructose.

Dietary macronutrient composition alters metabolism through several mechanisms, including post-translational modification (PTM) of proteins. To connect diet and molecular changes, here we performed short- and long-term feeding of mice with standard chow diet (SCD) and high-fat diet (HFD), with or without glucose or fructose supplementation, and quantified liver metabolites, 861 proteins, and 1,815 protein level-corrected mitochondrial acetylation and succinylation sites. Nearly half the acylation sites were altered by at least one diet; nutrient-specific changes in protein acylation sometimes encompass entire pathways. Although acetyl-CoA is an intermediate in both sugar and fat metabolism, acetyl-CoA had a dichotomous fate depending on its source; chronic feeding of dietary sugars induced protein hyperacetylation, whereas the same duration of HFD did not. Instead, HFD resulted in citrate accumulation, anaplerotic metabolism of amino acids, and protein hypo-succinylation. Together, our results demonstrate novel connections between dietary macronutrients, protein post-translational modifications, and regulation of fuel selection in liver.

Simultaneous Quantification of the Acetylome and Succinylome by 'One-Pot' Affinity Enrichment.

Protein posttranslational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi-PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here, a streamlined, quantitative label-free proteomic workflow-"one-pot" PTM enrichment-that enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein is described. Coupled with a label-free, data-independent acquisition (DIA), 2235 acetylated and 2173 succinylated peptides with the one-pot method are identified and quantified and peak areas are shown to be highly correlated between the one-pot and traditional single-PTM enrichments. The 'one-pot' method makes possible detection of multiple PTMs occurring on the same peptide, and it is shown that it can be used to make unique biological insights into PTM crosstalk. Compared to single-PTM enrichments, the one-pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue.

An acetylatable lysine controls CRP function in E. coli.

Transcriptional regulation is the key to ensuring that proteins are expressed at the proper time and the proper amount. In Escherichia coli, the transcription factor cAMP receptor protein (CRP) is responsible for much of this regulation. Questions remain, however, regarding the regulation of CRP activity itself. Here, we demonstrate that a lysine (K100) on the surface of CRP has a dual function: to promote CRP activity at Class II promoters, and to ensure proper CRP steady state levels. Both functions require the lysine's positive charge; intriguingly, the positive charge of K100 can be neutralized by acetylation using the central metabolite acetyl phosphate as the acetyl donor. We propose that CRP K100 acetylation could be a mechanism by which the cell downwardly tunes CRP-dependent Class II promoter activity, whilst elevating CRP steady state levels, thus indirectly increasing Class I promoter activity. This mechanism would operate under conditions that favor acetate fermentation, such as during growth on glucose as the sole carbon source or when carbon flux exceeds the capacity of the central metabolic pathways.

Characterization of Inner and Outer Membrane Proteins from Francisella tularensis Strains LVS and Schu S4 and Identification of Potential Subunit Vaccine Candidates.

Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies.IMPORTANCE The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make Francisella tularensis a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify F. tularensis as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States.

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry.

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories.

Identification and characterization of AckA-dependent protein acetylation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt) strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as "AckA-dependent". Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies focusing on specific acetylation sites that may have relevance in gonococcal pathogenesis and metabolism.

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro, succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5-/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5-/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5-/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function.

Generation of High-Quality SWATH® Acquisition Data for Label-free Quantitative Proteomics Studies Using TripleTOF® Mass Spectrometers.

Data-independent acquisition is a powerful mass spectrometry technique that enables comprehensive MS and MS/MS analysis of all detectable species, providing an information rich data file that can be mined deeply. Here, we describe how to acquire high-quality SWATH® Acquisition data to be used for large quantitative proteomic studies. We specifically focus on using variable sized Q1 windows for acquisition of MS/MS data for generating higher specificity quantitative data.

Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Response Pathway Genes skn-1, ire-1, and xbp-1.

Vitamin D has multiple roles, including the regulation of bone and calcium homeostasis. Deficiency of 25-hydroxyvitamin D, the major circulating form of vitamin D, is associated with an increased risk of age-related chronic diseases, including Alzheimer's disease, Parkinson's disease, cognitive impairment, and cancer. In this study, we utilized Caenorhabditis elegans to examine the mechanism by which vitamin D influences aging. We found that vitamin-D3-induced lifespan extension requires the stress response pathway genes skn-1, ire-1, and xbp-1. Vitamin D3 (D3) induced expression of SKN-1 target genes but not canonical targets of XBP-1. D3 suppressed an important molecular pathology of aging, that of widespread protein insolubility, and prevented toxicity caused by human ß-amyloid. Our observation that D3 improves protein homeostasis and slows aging highlights the importance of maintaining appropriate vitamin D serum levels and may explain why such a wide variety of human age-related diseases are associated with vitamin D deficiency.

Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH).

Post-translational modification of lysine residues by NƐ-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy. Graphical Abstract ᅟ.

Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4.

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level.