N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry.

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission.

Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric analysis of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry.

Proteomic analysis reveals virus-specific Hsp25 modulation in cardiac myocytes.

Viruses frequently infect the heart but clinical myocarditis is rare, suggesting that the cardiac antiviral response is uniquely effective. Indeed, the Type I interferon (IFN) response is cardiac cell-type specific and provides one integrated network of protection for the heart. Here, a proteomic approach was used to identify additional proteins that may be involved in the cardiac antiviral response.

Systematic comparison of the human saliva and plasma proteomes.

The proteome of human salivary fluid has the potential to open new doors for disease biomarker discovery. A recent study to comprehensively identify and catalog the human ductal salivary proteome led to the compilation of 1166 proteins. The protein complexity of both saliva and plasma is large, suggesting that a comparison of these two proteomes will provide valuable insight into their physiological significance and an understanding of the unique and overlapping disease diagnostic potential that each fluid provides.

Proteomics of Pyrococcus furiosus, a hyperthermophilic archaeon refractory to traditional methods.

Pyrococcus furiosus is one of the most extensively studied hyperthermophilic archaea. Proteins from this hyperthemophile organism are extremely thermostable and are highly resistant to chemical denaturants, organic solvents and proteolytic digestion. This thermostability makes it difficult to apply traditional methods of enzymatically digesting a complex mixture of proteins, commonly a first step in peptide generation in most shotgun proteomics methods.

Comprehensive defensin assay for saliva.

Defensins are highly basic cationic peptides that are important components of the innate and adaptive immune response pathways. In addition, these peptides are involved in CD8+ T cell response to HIV-1, increased pulmonary infection risk among cystic fibrosis patients, upregulated levels of HNP-5 for patients with ulcerative colitis and Crohn's disease, and monitoring HNP-3 levels as a tumor classification scheme for cutaneous T cell lymphomas, and have promise in the pharmaceutical field as a new class of antibiotics. Here we present a parallel assay for the alpha (HNP1-3) and beta (HBD1-2) classes of defensins in saliva that are naturally observed in the concentration range of 1 ng/mL to 10 microg/mL.

Automated proteomics of E. coli via top-down electron-transfer dissociation mass spectrometry.

Electron-transfer dissociation (ETD) has recently been introduced as a fragmentation method for peptide and protein analysis. Unlike collisionally induced dissociation (CID), fragmentation by ETD occurs randomly along the peptide backbone. With the use of the sequences determined from the protein termini and the parent protein mass, intact proteins can be unambiguously identified.

Minimizing back exchange in 18O/16O quantitative proteomics experiments by incorporation of immobilized trypsin into the initial digestion step.

Differential labeling of peptides via the use of the 18O-water proteolytic labeling method has been widely adopted for quantitative shotgun proteomics studies due to its simplicity and low reagent costs. In this report, the use of immobilized trypsin in the initial digestion step, in addition to the initial digestion step, is explored as a means to minimize postlabeling back exchange of 18O-labeled peptides into the 16O form when multidimensional peptide separation methods (here, isoelectric focusing of peptides) are incorporated into the sample workflow. Examples are shown with a mixture of standard proteins and a sample from an ongoing clinical proteomics study.

Immobilized pH gradient isoelectric focusing as a first-dimension separation in shotgun proteomics.

Shotgun proteomics, where a tryptic digest of a complex proteome sample is directly analyzed by either single dimensional or multidimensional liquid chromatography tandem mass spectrometry, has gained acceptance in the proteomics community at large and is widely used in core facilities. Here we review the development in our laboratory of an alternative first-dimension separation technique for shotgun proteomics, immobilized pH gradient isoelectric focusing (IPG-IEF). The key advantages of the technology over other multidimensional separation formats (simplicity, high resolution, and high sensitivity) are discussed.

A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics.

Recently, we have developed a high-resolution two-dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed-phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong-cation-exchange (SCX) chromatography in the first dimension.

Potential for false positive identifications from large databases through tandem mass spectrometry.

The biomedical research community at large is increasingly employing shotgun proteomics for large-scale identification of proteins from enzymatic digests. Typically, the approach used to identify proteins and peptides from tandem mass spectral data is based on the matching of experimentally generated tandem mass spectra to the theoretical best match from a protein database. Here, we present the potential difficulties of using such an approach without statistical consideration of the false positive rate, especially when large databases, as are encountered in eukaryotes are considered.

Gel based isoelectric focusing of peptides and the utility of isoelectric point in protein identification.

Here we present the theoretical and experimental evaluation of peptide isoelectric point as a method to aid in the identification of peptides from complex mixtures. Predicted pI values were found to match closely the experimentally obtained data, resulting in the development of a unique filter that lowers the effective false positive rate for peptide identification. Due to the reduction of the false positive rate, the cross-correlation parameters Xcorr and deltaCn from the SEQUEST program can be lowered resulting in 25% more peptide identifications.

Synthesis/degradation ratio mass spectrometry for measuring relative dynamic protein turnover.

One of the major unanswered questions in quantitative proteomics is that of dynamic protein turnover in the cell. Here we present a new approach to quantitative proteomics that measures the relative dynamic turnover of proteins in cellular systems. In this approach, termed synthesis/degradation ratio mass spectrometry, stable isotope labeling is employed to calculate a relative synthesis/degradation ratio that reflects the relative rate at which 13C is incorporated into individual proteins in the cell.

Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis.

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies.

Oligonucleotide mixture analysis via electrospray and ion/ion reactions in a quadrupole ion trap.

Electrospray ionization combined with ion/ion reactions in a quadrupole ion trap can be used for the direct analysis of oligonucleotide mixtures. Elements to the success of this approach include factors related to ionization, ion/ion reactions, and mass analysis. This paper deals with issues regarding the ion polarity combination, viz.

Ion/ion chemistry as a top-down approach for protein analysis.

Developing methodology for analyzing complex protein mixtures in a rapid fashion is one of the most challenging problems facing analytical biochemists today. Recent advances in mass spectrometry for the analysis of intact proteins (i.e.