Detecting Disulfide Bond Shuffling in Biologics Using Trypsin Platinum

Biologic therapeutics such as monoclonal antibodies and biosimilars are complex proteins that are susceptible to post-translational modifications (PTMs). These chemical modifications can affect the performance and activity of the biologic, potentially resulting in decreased potency and increased immunogenicity. Such modifications include glycosylation, deamidation, oxidation and disulfide bond shuffling. These PTMs can be signs of protein degradation, manufacturing issues or improper storage. Several of these modifications are well characterized, and methods exist for detecting them during biologic manufacture. However, disulfide shuffling is not particularly well characterized for biologics, and no methods exist to easily detect and quantify disulfide bond shuffling in biologics.

Disulfide bond shuffling occurs when the S-S linkage is not between a Cys and its normal partner
Disulfide bonds are important for protein conformation and function

Normally the cysteines in a protein will pair with a predictable or “normal” partner residue either within a polypeptide chain or between two polypeptide chains when they form disulfide bonds. These normal disulfide bonds are important for final protein conformation and stability. Indeed, disulfide bonds are considered an important quality indicator for biologics.

In a recently published study, Coghlan and colleagues designed a semi-automated method for characterizing disulfide bond shuffling on two IgG1 biologics: rituximab (originator drug Rituxan® and biosimilar Acellbia®) and bevacizumab (originator Avastin® and biosimilar Avegra®).

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How Can You Improve Protein Digests for Mass Spectrometry Analysis?

Can predigestion with trypisin (ribbon structure shown) improve protein digests for mass spectrometry analysis?
Can pre-digestion with trypsin improve mass spec analysis?

The trypsin protease cleaves proteins on the carboxyterminus of Arginine (Arg) and Lysine (Lys). This cleavage reaction leaves a positive charge on the C-terminus of the resulting peptide, which enhances mass spectrometry analysis (1,2). Because of this advantage, trypsin has become the most commonly used protease for mass spectrometry analysis. Other proteases which cleave diffrently from trypsin, yielding complementary data are also used in mass spec analysis: these include Asp-N and Glu-C , which cleave acidic residues, and chymotrypsin which cleaves at aromatic residues. The broad spectrum protease, proteinase K is also used for some proteomic analyses. In a recent study, Dau and colleagues investigated whether sequential digestion with trypsin followed by the complementary proteases could improve protein digests for mass spectrometry analysis.

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The Path is Clear: Trypsin Platinum is Here!

Mass spectrometry depends on the successful digestion of proteins using proteases. Many commercially available proteomic-grade trypsins contain natural contaminants that produce non-specific cleavages. Trypsin Platinum, a new protease from Promega provides maximum specificity, giving you cleaner and more conclusive data from mass spec.

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Mass Spec for Glycosylation Analysis of SARS-CoV-2 Proteins Implicated in Host-Cell Entry

The spike protein of the SARS-CoV-2 virus is a very commonly researched target in COVID-19 vaccine and therapeutic studies because it is an integral part of host cell entry through interactions between the S1 subunit of the spike protein with the ACE2 protein on the target cell surface. Viral proteins important in host cell entry are typically highly glycosylated. Looking at the sequence of the SARS-CoV-2 virus, researchers predict that the spike protein is highly glycosylated. In a recent study, researchers conducted a glycosylation analysis of SARS-CoV-2 proteins using mass spec analysis to determine the N-glycosylation profile of the subunits that make up the spike protein.

3d model of coronavirus covid-19 showing the spike protein. A recent study performed a glycosylation analysis of SARS-CoV-2 protein.

Glycans assist in protein folding and help the virus avoid immune recognition by the host. Glycosylation can also have an impact on the antigenicity of the virus, as well as potential effects on vaccine safety and efficacy. Mass spectrometry is widely used for viral characterization studies of influenza viruses. Specifically, mass spec has been used to study influenza protein glycosylation, antigen quantification, and determination of vaccine potency.

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More muscle from eggs? Proteo-lipid complex may help prevent age-associated loss of muscle-mass

In older people, low muscle mass is strongly associated with reduced functional capacity and an increased risk of disability. Myostatin is a negative regulator of muscle growth and has become an important target for pharmaceutical companies designing therapeutics to address age-associated muscle loss.

Anti-myostatin drugs increase muscle size and strength in preclinical studies. Fortetropin is a proteo-lipid complex made from fertilized egg yolk and shows anti-myostatin activity. When Fortetropin is provided as a supplement, lowered circulating myostatin levels are observed both in rodents and in young men. Fortetropin in combination with resistance exercise also lowers myostatin and increased lean body mass.

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Proteomics from a Different Point of View: Introducing ProAlanase, the Newest Mass-Spec Grade Protease from Promega

Sometimes, when using trypsin to study a protein sequence or protein modifications, sequence coverage just isn’t quite as complete as you’d like. Looking for a protease with novel cleavage specificity or a protease that functions well in a low pH environment? Promega has a protease for that.

ProAlanase is a new site-specific endoprotease that preferentially cleaves proteins on the C-terminal side of proline and alanine amino acids. The unique cleavage specificity of ProAlanase (also known as An-PEP or EndoPro; 1–3) can help to uncover parts of the proteome not previously accessible with proteases typically used in proteomic studies.

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Understanding the Structure of SARS-CoV-2 Spike Protein

Glycosylation is the process by which a carbohydrate is covalently attached totarget macromolecules, typically proteins. This modification serves various functions including guiding protein folding (1,2), promoting protein stability (2), and participating signaling functions (3).

ribbon structure of SARS-CoV-2 protein
Ribbon Structure of SARS-CoV-2 Spike Protein

SARS-CoV-2 utilizes an extensively glycosylated spike (S) protein that protrudes from the viral surface to bind to angiotensin-converting enzyme 2 (ACE2) to mediate host-cell entry. Vaccine development has been focused on this protein, which is the focus of the humoral immune response. Understanding the glycan structure of the SARS-CoV-2 virus spike (S) protein will be critical in the development of glycoprotine-based vaccine candidates.

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Bottom-up Proteomics: Need Help?

The use of mass spectrometry for the characterization of individual or complex protein samples continues to be one of the fastest growing fields in the life science market.

Bottom-up proteomics is the traditional approach to address these questions. Optimization of each the individual steps (e.g. sample prep, digestion and instrument performance) is critical to the overall success of the entire experiment.

To address issues that may arise in your experimental design, Promega has developed unique tools and complementary webinars to help you along the way.

Here you can find a summary of individual webinars for the following topics:

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Use of HIC high resolution chromatography and elastase for bottom up proteomics

One of the key applications used to characterize single or complex protein mixtures via bottom up proteomics is liquid chromatography−tandem mass spectrometry (LC−MS/MS).
Recent technical advances allow for identification of >10 000 proteins in a cancer cell line. On the peptide level chromatography methods, like strong cation exchange (SCX)
and hydrophilic interaction chromatography (HILIC), as well as high-pH reversed phase chromatography have been employed successfully. Because of its robustness
and ease of handling, the classical and still widely used approach for protein fractionation prior to LC− MS/MS is gel-based separation under denaturing conditions (SDS-PAGE).
Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used
to study post-translational modifications of proteins and drug−protein interactions.  HIC is a high-resolution chromatography mode based on the interaction of
weakly hydrophobic ligands of the stationary phase with hydrophobic patches on the surface of the tertiary structure of proteins. By employment of high concentrations
of structure-promoting (“kosmotropic”) salts, proteins in HIC retain their conform

In a recent publication, HIC was used to separate proteins, followed by bottom up LC−MS/MS experiments (1).  HIC was used to fractionate antibody species
followed by comprehensive peptide mapping as well as to study protein complexes in human cells. The results indicated that HIC−reversed-phase chromatography (RPC)
mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

An additional observation noted that tryptic digests of the antibody used in the study yielded a protein coverage of 56% for the light chain and 63.2% for the
heavy chain. A consecutive proteolytic digestion protocol combing on-filter trypsin and elastase digestion drastically improved sequence coverage of
both light (100%) and heavy chains (99.2%).

1. Rackiewicz, M. et al. (2017) Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes. J.Proteome.Res. 16, 2318–23.

Optimization of Alternative Proteases for Bottom-Up Proteomics

Alternate Proteases CoverBottom-up proteomics focuses on the analysis of protein mixtures after enzymatic digestion of the proteins into peptides. The resulting complex mixture of peptides is analyzed by reverse-phase liquid chromatography (RP-LC) coupled to tandem mass spectrometry (MS/MS). Identification of peptides and subsequently proteins is completed by matching peptide fragment ion spectra to theoretical spectra generated from protein databases.

Trypsin has become the gold standard for protein digestion to peptides for shotgun proteomics. Trypsin is a serine protease. It cleaves proteins into peptides with an average size of 700-1500 daltons, which is in the ideal range for MS (1). It is highly specific, cutting at the carboxyl side of arginine and lysine residues. The C-terminal arginine and lysine peptides are charged, making them detectable by MS. Trypsin is highly active and tolerant of many additives.

Even with these technical features, the use of trypsin in bottom-up proteomics may impose certain limits in the ability to grasp the full proteome, Tightly-folded proteins can resist trypsin digestion. Post-translational modifications (PTMs) present a different challenge for trypsin because glycans often limit trypsin access to cleavage sites, and acetylation makes lysine and arginine residues resistant to trypsin digestion.

To overcome these problems, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated when using these alternative proteases have not been systematically documented.

In a recent reference (2), optimized protocols for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, Lys-C, Lys-N, Asp-N, Glu-C and Arg-C have been created.

Data describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in approximately in 2 days.


  1. Laskay, U. et al. (2013) Proteome Digestion Specificity Analysis for the Rational Design of Extended Bottom-up and middle-down proteomics experiments. J of Proteome Res. 12, 5558–69.
  2. Giansanti, P. et. al. (2016) Six alternative protease for mass spectrometry based proteomics beyond trypsin. Nat. Protocols 11, 993–6