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.

Facile Preparation of Peptides for Mass Spectrometry Analysis in Bottom-Up Proteomics Workflows.

Mass spectrometry (MS) is routinely used to identify, characterize, and quantify biological molecules. For protein analysis, MS-based workflows can be broadly categorized as top-down or bottom-up, depending on whether the proteins are analyzed as intact molecules or first digested into peptides. This article outlines steps for preparing peptide samples for MS as part of a bottom-up proteomics workflow, providing versatile methods suitable for discovery and targeted analyses in qualitative and quantitative workflows. Resulting samples contain peptides of suitable size for analysis by MS instrumentation generally available to modern research laboratories, including MS coupled to either liquid chromatography (LC) or matrix-assisted laser desorption/ionization (MALDI) interfaces. This article incorporates recent developments in methodologies and consumables to facilitate sample preparation. The protocols are well-suited to users without prior experience in proteomics and include methods for universally applicable suspension trap processing and for alternate in-solution processing to accommodate a range of sample types. Cleanup, quantification, and fractionation procedures are also described. © 2021 The Authors. Basic Protocol: Preparation of high-complexity peptide samples for mass spectrometry analysis using S-Trap™ processing Alternate Protocol 1: Preparation of low- to moderate-complexity peptide samples for mass spectrometry analysis using in-solution processing Alternate Protocol 2: Detergent, polymer, and salt removal from peptide samples before mass spectrometry analysis using SP2 processing Support Protocol 1: Protein quantification using Pierce 660 nm assay Support Protocol 2: Peptide quantification using Pierce quantitative fluorometric peptide assay Support Protocol 3: High-pH fractionation of complex peptide samples.

Parallel Determination of Polypeptide and Oligosaccharide Connectivities by Energy-Resolved Collison-Induced Dissociation of Protonated O-Glycopeptides Derived from Nonspecific Proteolysis.

Collision-induced dissociation (CID) is by far the most broadly applied dissociation method used for tandem mass spectrometry (MS/MS). This includes MS/MS-based structural interrogation of glycopeptides for applications in glycoproteomics. The end goal of such measurements is to determine the monosaccharide connectivity of the glycan, the amino acid sequence of the peptide, and the site of glycosylation for each glycopeptide of interest. In turn, this allows inferences with respect to the glycoprofile of the intact glycoprotein. For glycopeptide analysis, CID is best known for the ability to determine glycosidic topology of the oligosaccharide group; however, CID has also been shown to produce amide bond cleavage of the polypeptide group. Whether structural information is obtained for the glycan or the peptide has been found to depend on the applied collision energy. While these energy-resolved fragmentation pathways have been the subject of several studies on N-linked glycopeptides, there remains a dearth of similar work on O-linked glycopeptides. In this study, MS/MS via CID was shown to provide substantial peptide backbone fragmentation, in addition to glycosidic fragmentation, in an energy-dependent manner. While qualitatively similar to previous findings for N-glycopeptides, the energy-resolved CID (ER-CID) of O-glycopeptides was found to be substantially more sensitive to the collision energy setting. Thus, deliberately obtaining either glycan or peptide dissociation is a more delicate undertaking for O-glycopeptides. Establishing a more complete understanding of O-glycopeptide ER-CID is likely to have a substantive impact on how O-glycoproteomic analysis is approached in the future.

A real-time, fluorescence-based assay for Rho-associated protein kinase activity.

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z'-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC50 values of 3.6 µM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.