Comprehensive Prostate Fluid-Based Spectral Libraries for Enhanced Protein Detection in Urine.

Biofluids contain molecules in circulation and from nearby organs that can be indicative of disease states. Characterizing the proteome of biofluids with DIA-MS is an emerging area of interest for biomarker discovery; yet, there is limited consensus on DIA-MS data analysis approaches for analyzing large numbers of biofluids. To evaluate various DIA-MS workflows, we collected urine from a clinically heterogeneous cohort of prostate cancer patients and acquired data in DDA and DIA scan modes. We then searched the DIA data against urine spectral libraries generated using common library generation approaches or a library-free method. We show that DIA-MS doubles the sample throughput compared to standard DDA-MS with minimal losses to peptide detection. We further demonstrate that using a sample-specific spectral library generated from individual urines maximizes peptide detection compared to a library-free approach, a pan-human library, or libraries generated from pooled, fractionated urines. Adding urine subproteomes, such as the urinary extracellular vesicular proteome, to the urine spectral library further improves the detection of prostate proteins in unfractionated urine. Altogether, we present an optimized DIA-MS workflow and provide several high-quality, comprehensive prostate cancer urine spectral libraries that can streamline future biomarker discovery studies of prostate cancer using DIA-MS.

Importance of evaluating protein glycosylation in pluripotent stem cell-derived cardiomyocytes for research and clinical applications.

Proper protein glycosylation is critical to normal cardiomyocyte physiology. Aberrant glycosylation can alter protein localization, structure, drug interactions, and cellular function. The in vitro differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM) has become increasingly important to the study of protein function and to the fields of cardiac disease modeling, drug testing, drug discovery, and regenerative medicine. Here, we offer our perspective on the importance of protein glycosylation in hPSC-CM. Protein glycosylation is dynamic in hPSC-CM, but the timing and extent of glycosylation are still poorly defined. We provide new data highlighting how observed changes in hPSC-CM glycosylation may be caused by underlying differences in the protein or transcript abundance of enzymes involved in building and trimming the glycan structures or glycoprotein gene products. We also provide evidence that alternative splicing results in altered sites of glycosylation within the protein sequence. Our findings suggest the need to precisely define protein glycosylation events that may have a critical impact on the function and maturation state of hPSC-CM. Finally, we provide an overview of analytical strategies available for studying protein glycosylation and identify opportunities for the development of new bioinformatic approaches to integrate diverse protein glycosylation data types. We predict that these tools will promote the accurate assessment of protein glycosylation in future studies of hPSC-CM that will ultimately be of significant experimental and clinical benefit.

SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates.

MOTIVATION: Cell-type-specific surface proteins can be exploited as valuable markers for a range of applications including immunophenotyping live cells, targeted drug delivery and in vivo imaging. Despite their utility and relevance, the unique combination of molecules present at the cell surface are not yet described for most cell types. A significant challenge in analyzing 'omic' discovery datasets is the selection of candidate markers that are most applicable for downstream applications. RESULTS: Here, we developed GenieScore, a prioritization metric that integrates a consensus-based prediction of cell surface localization with user-input data to rank-order candidate cell-type-specific surface markers. In this report, we demonstrate the utility of GenieScore for analyzing human and rodent data from proteomic and transcriptomic experiments in the areas of cancer, stem cell and islet biology. We also demonstrate that permutations of GenieScore, termed IsoGenieScore and OmniGenieScore, can efficiently prioritize co-expressed and intracellular cell-type-specific markers, respectively. AVAILABILITY AND IMPLEMENTATION: Calculation of GenieScores and lookup of SPC scores is made freely accessible via the SurfaceGenie web application: www.cellsurfer.net/surfacegenie. CONTACT: Rebekah.gundry@unmc.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes.

Cell surface glycoproteins play critical roles in maintaining cardiac structure and function in health and disease and the glycan-moiety attached to the protein is critical for proper protein folding, stability and signaling [1]. However, despite mounting evidence that glycan structures are key modulators of heart function and must be considered when developing cardiac biomarkers, we currently do not have a comprehensive view of the glycans present in the normal human heart. In the current study, we used porous graphitized carbon liquid chromatography interfaced with mass spectrometry (PGC-LC-MS) to generate glycan structure libraries for primary human heart tissue homogenate, cardiomyocytes (CM) enriched from human heart tissue, and human induced pluripotent stem cell derived CM (hiPSC-CM). Altogether, we established the first reference structure libraries of the cardiac glycome containing 265 N- and O-glycans. Comparing the N-glycome of CM enriched from primary heart tissue to that of heart tissue homogenate, the same pool of N-glycan structures was detected in each sample type but the relative signal of 21 structures significantly differed between samples, with the high mannose class increased in enriched CM. Moreover, by comparing primary CM to hiPSC-CM collected during 20-100 days of differentiation, dynamic changes in the glycan profile throughout in vitro differentiation were observed and differences between primary and hiPSC-CM were revealed. Namely, >30% of the N-glycome significantly changed across these time-points of differentiation and only 23% of the N-glycan structures were shared between hiPSC-CM and primary CM. These observations are an important complement to current genomic, transcriptomic, and proteomic profiling and reveal new considerations for the use and interpretation of hiPSC-CM models for studies of human development, disease, and drug testing. Finally, these data are expected to support future regenerative medicine efforts by informing targets for evaluating the immunogenic potential of hiPSC-CM and harnessing differences between immature, proliferative hiPSC-CM and adult primary CM.

Addressing Cellular Heterogeneity in Cancer through Precision Proteomics.

Cells exhibit a broad spectrum of functions driven by differences in molecular phenotype. Understanding the heterogeneity between and within cell types has led to advances in our ability to diagnose and manipulate biological systems. Heterogeneity within and between tumors still poses a challenge to the development and efficacy of therapeutics. In this Perspective we review the toolkit of protein-level experimental approaches for investigating cellular heterogeneity. We describe how innovative approaches and technical developments have supported the advent of bottom-up single-cell proteomic analysis and present opportunities and challenges within cancer research. Finally, we introduce the concept of "precision proteomics" and discuss how the advantages and limitations of various experimental approaches render them suitable for different biological systems and questions.

SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic Analyses.

Peptide cleanup is essential for the removal of contaminating substances that may be introduced during sample preparation steps in bottom-up proteomic workflows. Recent studies have described benefits of carboxylate-modified paramagnetic particles over traditional reversed-phase methods for detergent and polymer removal, but challenges with reproducibility have limited the widespread implementation of this approach among laboratories. To overcome these challenges, the current study systematically evaluated key experimental parameters regarding the use of carboxylate-modified paramagnetic particles and determined those that are critical for maximum performance and peptide recovery and those for which the protocol is tolerant to deviation. These results supported the development of a detailed, easy-to-use standard operating protocol, termed SP2, which can be applied to remove detergents and polymers from peptide samples while concentrating the sample in solvent that is directly compatible with typical LC-MS workflows. We demonstrate that SP2 can be applied to phosphopeptides and glycopeptides and that the approach is compatible with robotic liquid handling for automated sample processing. Altogether, the results of this study and accompanying detailed operating protocols for both manual and automated processing are expected to facilitate reproducible implementation of SP2 for various proteomics applications and will especially benefit core or shared resource facilities where unknown or unexpected contaminants may be particularly problematic.

A call to adopt a "fit for purpose" approach to antibody validation for flow cytometry analyses of stem cell models and beyond.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) can be exploited as models for a wide range of research applications and numerous protocols for generating hPSC-CMs have been described. However, it is currently not possible to direct differentiation to a single, homogeneous end point, and the resulting heterogeneity may be variable among laboratories, cell lines, and protocols. Consequently, the ability to assess phenotypic heterogeneity of the cell population is critical to the interpretation, repeatability, and reproduction of hPSC-CM studies. While flow cytometry is well suited for this purpose, a review of published literature reveals there is currently no consensus regarding which marker, antibody, or protocol is best suited to enable comparisons of hPSC-CM culture heterogeneity. Moreover, the lack of available experimental detail, combined with the variability in the approaches used for hPSC-CM evaluation, makes it challenging to reproduce, interpret, and compare published data. Consequently, this article calls for an alignment of the way researchers approach the routine use and documentation of the antibodies and controls used during flow cytometry-based assessment of hPSC-CM cultures. We advocate for the adoption of a "fit for purpose" validation mindset, whereby antibodies and experimental conditions are demonstrated as specific within a defined experimental design and biological context. Overall, we expect that by adhering to rigorous standards for antibody validation and use, reporting of experimental details, and presentation of data, the concepts emphasized here will promote enhanced utility and dialogue regarding hPSC-CM for a variety of research and translational applications by enabling more accurate comparisons of results among studies.

Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin.

Mechanotransduction refers to the processes whereby mechanical stimuli are converted into electrochemical signals that allow for the sensation of our surrounding environment through touch. Despite its fundamental role in our daily lives, the molecular and cellular mechanisms of mechanotransduction are not yet well-defined. Previous data suggest that keratinocytes may release factors that activate or modulate cutaneous sensory neuron terminals, including small molecules, lipids, peptides, proteins, and oligosaccharides. This study presents a first step toward identifying soluble mediators of keratinocyte-sensory neuron communication by evaluating the potential for top-down mass spectrometry to identify proteoforms released during 1 min of mechanical stimulation of mouse skin from naïve animals. Overall, this study identified 47 proteoforms in the secretome of mouse hind paw skin, of which 14 were differentially released during mechanical stimulation, and includes proteins with known and previously unknown relevance to mechanotransduction. Finally, this study outlines a bioinformatic workflow that merges output from two complementary analysis platforms for top-down data and demonstrates the utility of this workflow for integrating quantitative and qualitative data.

N-glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm.

Using cell surface capture technology, the cell surface N-glycoproteome of human-induced pluripotent stem cell derived hepatic endoderm cells was assessed. Altogether, 395 cell surface N-glycoproteins were identified, represented by 1273 N-glycopeptides. This study identified N-glycoproteins that are not predicted to be localized to the cell surface and provides experimental data that assist in resolving ambiguous or incorrectly annotated transmembrane topology annotations. In a proof-of-concept analysis, combining these data with other cell surface proteome datasets is useful for identifying potentially cell type and lineage restricted markers and drug targets to advance the use of stem cell technologies for mechanistic developmental studies, disease modeling, drug discovery, and regenerative medicine.

Cell Surface Proteomics of N-Linked Glycoproteins for Typing of Human Lymphocytes.

Lymphocytes are immune cells that are critical for the maintenance of adaptive immunity. Differentiation of lymphoid progenitors yields B-, T-, and NK-cell subtypes that individually correlate with specific forms of leukemia or lymphoma. Therefore, it is imperative a precise method of cell categorization is utilized to detect differences in distinct disease states present in patients. One viable means of classification involves evaluation of the cell surface proteome of lymphoid malignancies. Specifically, this manuscript details the use of an antibody independent approach known as Cell Surface Capture Technology, to assess the N-glycoproteome of four human lymphocyte cell lines. Altogether, 404 cell surface N-glycoproteins were identified as markers for specific cell types involved in lymphocytic malignancies, including 82 N-glycoproteins that had not been previously been described for B or T cells within the Cell Surface Protein Atlas. Comparative analysis, hierarchical clustering techniques, and label-free quantitation were used to reveal proteins most informative for each cell type. Undoubtedly, the characterization of the cell surface proteome of lymphoid malignancies is a first step toward improving personalized diagnosis and treatment of leukemia and lymphoma.

Transcriptional inhibition of etv2 expression is essential for embryonic cardiac development.

E-twenty six variant 2 (Etv2) transcription factor participates in cardiac, vascular-endothelial and blood cell lineage specification decisions during embryonic development. Previous studies have identified genomic elements in the etv2 locus responsible for vascular endothelial cell specification. Using transgenic analysis in zebrafish, we report here an etv2 proximal promoter fragment that prevents transgene misexpression in myocardial progenitor cells. This inhibition of etv2 expression in the cardiac progenitor population is partly mediated by Scl and Nkx2.5, likely through direct binding to the etv2 promoter, and cis-regulatory elements located in the first and second introns. The results identify an etv2 cis-regulatory mechanism controlling cardiovascular fate choice implying that etv2 participates in a transcriptional network mediating developmental plasticity of endothelial progenitor cells during embryonic development.

Combine and conquer: surfactants, solvents, and chaotropes for robust mass spectrometry based analyses of membrane proteins.

Mass spectrometry (MS) based proteomic technologies enable the identification and quantification of membrane proteins as well as their post-translational modifications. A prerequisite for their quantitative and reliable MS-based bottom-up analysis is the efficient digestion into peptides by proteases, though digestion of membrane proteins is typically challenging due to their inherent properties such as hydrophobicity. Here, we investigated the effect of eight commercially available MS-compatible surfactants, two organic solvents, and two chaotropes on the enzymatic digestion efficiency of membrane protein-enriched complex mixtures in a multiphase study using a gelfree approach. Multiple parameters, including the number of peptides and proteins identified, total protein sequence coverage, and digestion specificity were used to evaluate transmembrane protein digestion performance. A new open-source software tool was developed to allow for the specific assessment of transmembrane domain sequence coverage. Results demonstrate that while Progenta anionic surfactants outperform other surfactants when tested alone, combinations of guanidine and acetonitrile improve performance of all surfactants to near similar levels as well as enhance trypsin specificity to >90%, which has critical implications for future quantitative and qualitative proteomic studies.

Organelle resolved proteomics uncovers PLA2R1 as a novel cell surface marker required for chordoma growth.

Chordomas are clinically aggressive tumors with a high rate of disease progression despite maximal therapy. Given the limited therapeutic options available, there remains an urgent need for the development of novel therapies to improve clinical outcomes. Cell surface proteins are attractive therapeutic targets yet are challenging to profile with common methods. Four chordoma cell lines were analyzed by quantitative proteomics using a differential ultracentrifugation organellar fractionation approach. A subtractive proteomics strategy was applied to select proteins that are plasma membrane enriched. Systematic data integration prioritized PLA2R1 (secretory phospholipase A2 receptor-PLA2R1) as a chordoma-enriched surface protein. The expression profile of PLA2R1 was validated across chordoma cell lines, patient surgical tissue samples, and normal tissue lysates via immunoblotting. PLA2R1 expression was further validated by immunohistochemical analysis in a richly annotated cohort of 25-patient tissues. Immunohistochemistry analysis revealed that elevated expression of PLA2R1 is correlated with poor prognosis. Using siRNA- and CRISPR/Cas9-mediated knockdown of PLA2R1, we demonstrated significant inhibition of 2D, 3D and in vivo chordoma growth. PLA2R1 depletion resulted in cell cycle defects and metabolic rewiring via the MAPK signaling pathway, suggesting that PLA2R1 plays an essential role in chordoma biology. We have characterized the proteome of four chordoma cell lines and uncovered PLA2R1 as a novel cell-surface protein required for chordoma cell survival and association with patient outcome.

Droplet-based proteomics reveals CD36 as a marker for progenitors in mammary basal epithelium.

Deep proteomic profiling of rare cell populations has been constrained by sample input requirements. Here, we present DROPPS (droplet-based one-pot preparation for proteomic samples), an accessible low-input platform that generates high-fidelity proteomic profiles of 100-2,500 cells. By applying DROPPS within the mammary epithelium, we elucidated the connection between mitochondrial activity and clonogenicity, identifying CD36 as a marker of progenitor capacity in the basal cell compartment. We anticipate that DROPPS will accelerate biology-driven proteomic research for a multitude of rare cell populations.

Prostate cancer reshapes the secreted and extracellular vesicle urinary proteomes.

Urine is a complex biofluid that reflects both overall physiologic state and the state of the genitourinary tissues through which it passes. It contains both secreted proteins and proteins encapsulated in tissue-derived extracellular vesicles (EVs). To understand the population variability and clinical utility of urine, we quantified the secreted and EV proteomes from 190 men, including a subset with prostate cancer. We demonstrate that a simple protocol enriches prostatic proteins in urine. Secreted and EV proteins arise from different subcellular compartments. Urinary EVs are faithful surrogates of tissue proteomes, but secreted proteins in urine or cell line EVs are not. The urinary proteome is longitudinally stable over several years. It can accurately and non-invasively distinguish malignant from benign prostatic lesions and can risk-stratify prostate tumors. This resource quantifies the complexity of the urinary proteome and reveals the synergistic value of secreted and EV proteomes for translational and biomarker studies.

Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts.

Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging.

Prostate Cancer Reshapes the Secreted and Extracellular Vesicle Urinary Proteomes.

Urine is a complex biofluid that reflects both overall physiologic state and the state of the genitourinary tissues through which it passes. It contains both secreted proteins and proteins encapsulated in tissue-derived extracellular vesicles (EVs). To understand the population variability and clinical utility of urine, we quantified the secreted and EV proteomes from 190 men, including a subset with prostate cancer. We demonstrate that a simple protocol enriches prostatic proteins in urine. Secreted and EV proteins arise from different subcellular compartments. Urinary EVs are faithful surrogates of tissue proteomes, but secreted proteins in urine or cell line EVs are not. The urinary proteome is longitudinally stable over several years. It can accurately and non-invasively distinguish malignant from benign prostatic lesions, and can risk-stratify prostate tumors. This resource quantifies the complexity of the urinary proteome, and reveals the synergistic value of secreted and EV proteomes for translational and biomarker studies.

Differential DNA damage repair and PARP inhibitor vulnerability of the mammary epithelial lineages.

It is widely assumed that all normal somatic cells can equally perform homologous recombination (HR) and non-homologous end joining in the DNA damage response (DDR). Here, we show that the DDR in normal mammary gland inherently depends on the epithelial cell lineage identity. Bioinformatics, post-irradiation DNA damage repair kinetics, and clonogenic assays demonstrated luminal lineage exhibiting a more pronounced DDR and HR repair compared to the basal lineage. Consequently, basal progenitors were far more sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis) in both mouse and human mammary epithelium. Furthermore, PARPi sensitivity of murine and human breast cancer cell lines as well as patient-derived xenografts correlated with their molecular resemblance to the mammary progenitor lineages. Thus, mammary epithelial cells are intrinsically divergent in their DNA damage repair capacity and PARPi vulnerability, potentially influencing the clinical utility of this targeted therapy.

CIRFESS: An Interactive Resource for Querying the Set of Theoretically Detectable Peptides for Cell Surface and Extracellular Enrichment Proteomic Studies.

Cell surface transmembrane, extracellular, and secreted proteins are high value targets for immunophenotyping, drug development, and studies related to intercellular communication in health and disease. As the number of specific and validated affinity reagents that target this subproteome are limited, mass spectrometry (MS)-based approaches will continue to play a critical role in enabling discovery and quantitation of these molecules. Given the technical considerations that make MS-based cell surface proteome studies uniquely challenging, it can be difficult to select an appropriate experimental approach. To this end, we have integrated multiple prediction strategies and annotations into a single online resource, Compiled Interactive Resource for Extracellular and Surface Studies (CIRFESS). CIRFESS enables rapid interrogation of the human proteome to reveal the cell surface proteome theoretically detectable by current approaches and highlights where current prediction strategies provide concordant and discordant information. We applied CIRFESS to identify the percentage of various subsets of the proteome which are expected to be captured by targeted enrichment strategies, including two established methods and one that is possible but not yet demonstrated. These results will inform the selection of available proteomic strategies and development of new strategies to enhance coverage of the cell surface and extracellular proteome. CIRFESS is available at www.cellsurfer.net/cirfess.