Genomics research shows that more than a million proteins are encoded by approximately 30,000 human genes. Proteomics, the study of proteins encoded by the genome, includes identifying post-translational modifications, structural analyses, protein localization studies, and protein quantitation. Mass spectroscopybased techniques have evolved as a powerful tool in proteomics. Stable isotope-labeled peptides (SIL peptides) are chemically and physically indistinguishable from their endogenous counterparts concerning retention time, ionization efficiency, and fragmentation pathways.

Peptides play a vital role in the pharmaceutical industry and drug therapeutic development; however, their in vivo applications are sometimes limited due to fast degradation by proteases, poor solubility, antigenic responses, and glomerular filtration in the kidney. The covalent attachment of polyethylene glycol (PEG) chains to peptides is one approach that can reduce immunogenicity, improve solubility, and reduce renal clearance.

Hydrocarbon-stapled peptides are locked into their bioactive alpha-helical conformation through the site-specific introduction of a chemical brace, an all-hydrocarbon staple. The idea of peptide stapling was introduced to overcome the limitations of two broad classes of therapeutic agents (small molecules and protein biologics) in targeting intracellular protein-protein interactions. Small molecules only work on proteins with a specific surface feature, and most protein biologics do not penetrate cells. Because stapled peptides are locked into a stabilized α-helical structure (the most common element of protein secondary structures), they can easily penetrate cells.

Protein-protein and protein-peptide interactions play critical roles in all types of cellular processing. Peptides are natural partners to proteins and, as ligands, bind to proteins with high affinity due to their capacity to adapt to the often flexible protein surface. Despite this, peptides have drawbacks as drug candidates that include low plasma bioavailability, instability from proteolytic enzymes, and poor passive membrane permeability. Some success has been achieved with linear peptides, particularly peptides that maintain α-helical secondary structures. These motifs can be introduced to stabilized α-helical motifs by common “peptide-stapling” approaches, but stapled peptides can suffer from low bioactivity and poor solubility. Another strategy to maintain peptide secondary structure is modification by macrocyclization.

Mutations to the RBD of SARS-CoV have resulted in the reorganization of SARS-CoV-2 RBD, specifically to the loops that are in close proximity to the ACE2-binding ridge. Due to these structural changes, an additional intramolecular main-chain hydrogen bond is gained between Asn487 and Ala475 (Figure 4a). The Asn487–Ala475 hydrogen bond causes the RBM of SARS-CoV-2 to be more structurally compact, enabling the loop containing Ala475 to move into closer proximity to the ACE2 α1 helix. Closer contact between the RBM and α1 helix of ACE2 results in more intermolecular interactions (Figure 4a,b). Some of these interactions include hydrogen bonds between Gln493 (SARS-CoV-2) and Glu35 (ACE2),[3b,c] and main-chain hydrogen bonds between Gly502 and Lys31.

This guide will help you understand how to read ESI spectra for large and complex bio-molecules like peptides. Other techniques such as high-performance liquid chromatography (HPLC) show one major peak (i.e., absorbance and purity for RP-HPLC) in the spectra, making the interpretation straightforward. ESI-MS spectra are more complicated due to the multiplicity of the data. While most small molecules (100-500 AMU) behave well in ESI-MS and ionize as a single charged state, larger biomolecules, such as peptides, ionize as multiple charged variants. A major advantage of ESI-MS over other mass spectroscopy techniques is that molecular fragmentation is rare (t-Boc groups and a few other protection groups are an exception), which is why ESI-MS is often referred to as a soft ionization technique.

Solid-phase peptide synthesis (SPPS) has many advantages over liquid-phase peptide synthesis (LPPS) for preparing and manufacturing synthetic peptides. Except the synthesis of short peptide sequences (i.e., less than five amino acid residues), SPPS is faster, more efficient, and more economical than liquid-phase peptide synthesis (LPPS). Some of the advantages of SPPS include: (1) Excess reagents and products can be easily washed away, (2) using excess reagents to increase reaction rates and drive reactions to completion, (3) intermediates do not require isolation or characterization, (4) access to a broader range of solvents with low volatility and high polarity, (5) tethered peptide provides a ‘pseudo-dilute’ microenvironment, which can inhibit intermolecular reactions, making some modifications easier to accomplish, and (6) compatibility with automated synthesis technology.