Veneer Is a Webtool for Rapid, Standardized, and Transparent Interpretation, Annotation, and Reporting of Mammalian Cell Surface N-Glycocapture Data.

Currently, no consensus exists regarding criteria required to designate a protein within a proteomic data set as a cell surface protein. Most published proteomic studies rely on varied ontology annotations or computational predictions instead of experimental evidence when attributing protein localization. Consequently, standardized approaches for analyzing and reporting cell surface proteome data sets would increase confidence in localization claims and promote data use by other researchers. Recently, we developed Veneer, a web-based bioinformatic tool that analyzes results from cell surface N-glycocapture workflows─the most popular cell surface proteomics method used to date that generates experimental evidence of subcellular location. Veneer assigns protein localization based on defined experimental and bioinformatic evidence. In this study, we updated the criteria and process for assigning protein localization and added new functionality to Veneer. Results of Veneer analysis of 587 cell surface N-glycocapture data sets from 32 published studies demonstrate the importance of applying defined criteria when analyzing cell surface proteomics data sets and exemplify how Veneer can be used to assess experimental quality and facilitate data extraction for informing future biological studies and annotating public repositories.

The GENTIL Method for Isolation of Human Adult Cardiomyocytes from Cryopreserved Tissue for Proteomic Analyses.

Heart failure is a serious clinical and economic health care problem, and its clinical progression is linked to pathological cardiac remodeling. Due to the heterogeneity of heart failure, lack of animal models to accurately represent advanced heart failure, and limited access to fresh human cardiac tissue, little is known regarding cell-type-specific mechanisms and context-specific functions of cardiomyocytes during disease development processes. While mass spectrometry has been increasingly applied to unravel changes in the proteome associated with cardiovascular physiology and disease, most studies have used homogenized tissue. Therefore, new studies using isolated cardiomyocytes are necessary to gain a better understanding of the intricate cell-type-specific molecular mechanisms underlying the pathophysiology of heart failure. This chapter describes the GENTIL method, which incorporates recent technological developments in sample handling, for isolation of cardiomyocytes from cryopreserved human cardiac tissues for use in proteomic analyses.

Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes.

The heart is composed of multiple cell types, each with a specific function. Cell-type-specific approaches are necessary for defining the intricate molecular mechanisms underlying cardiac development, homeostasis, and pathology. While single-cell RNA-seq studies are beginning to define the chamber-specific cellular composition of the heart, our views of the proteome are more limited because most proteomics studies have utilized homogenized human cardiac tissue. To promote future cell-type specific analyses of the human heart, we describe the first method for cardiomyocyte isolation from cryopreserved human cardiac tissue followed by flow cytometry for purity assessment. We also describe a facile method for preparing isolated cardiomyocytes and whole cardiac tissue homogenate for bottom-up proteomic analyses. Prior experience in dissociating cardiac tissue or proteomics is not required to execute these methods. We compare different sample preparation workflows and analysis methods to demonstrate how these can impact the depth of proteome coverage achieved. We expect this how-to guide will serve as a starting point for investigators interested in general and cell-type-specific views of the cardiac proteome.