NovaBioAssays offers various methods to determine protein molecular weights, including the analysis of intact proteins under native or denaturing conditions, the use of enzymes and/or reducing conditions to deliver subunit-level mass analysis, employing liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS), such as Bruker QToF, Thermo Q Exactive and Q Exactive Plus, all coupled with Waters UPLCs.
This method measures the molecular weight of a protein molecule. The protein sample is desalted on a short reverse phase column and analyzed on a High Resolution mass spectrometer. The protein picks up multiple charges during electrospray and forms a so-called charged envelope. A maximum entropy algorithm is applied to deconvolute the multiple charged mass peaks into the molecular mass of the original protein. Comparing the measured mass with the predicted value from the protein sequence can confirm the sequence identity. Our method can readily measure proteins up to 300 KDa in size and routinely achieves mass accuracy within +/- 1 Da for protein around 50 KDa, which is sufficient to detect the majority of point mutations and post-translational modifications. Many therapeutic proteins (e.g., IgG) are glycosylated and the mass spectra of these proteins will have branching patterns resulted from the heterogeneity in glycan structures. Our method can resolve such heterogeneity of proteins with up to two glycosylation sites. When the protein has multiple glycosylation sites, the pattern becomes overly crowded and the mass for individual glycoform can no longer be determined. For such proteins, deglycosylation may be necessary to simplify the spectrum profile.
Peptide mapping is essential for characterization and elucidation of the primary amino acid structure of proteins. First the protein is digested into its constituent peptides commonly via enzymatic digestion. The resulting peptides are separated by HPLC with detection by MS or MS/MS, for proof of identity, sequence confirmation and identification of any modifications, and quantitation, if appropriate, including disulfide linkages, glycosylation, oxidation, deamidation, phosphorylation and integrity of N- and C-termini, etc.
Structural characterization at peptide mapping level highlights the identification of post translational modifications (PTMs), such as disulfide linkages, glycosylation, oxidation, deamidation, phosphorylation and integrity of N- and C-termini, etc, and quantitation, if appropriate.
We have extensive expertise in analyzing modifications including:
Depending on the nature of the modifications we adopt different approaches in sample preparation and mass spectrometric data collection. For stoichiometric modifications such as PEGylation and drug conjugation, where the majority of the protein is expected to be modified, we start with intact mass analysis to capture the global profile of the molecular species and then followed by peptide mapping. For low abundant modifications, we rely on specific enrichment techniques to harvest the modified species, i.e. metal affinity for phosphopeptides. We also take advantage of specific fragmentation signals from modified peptides, i.e., loss of phosphoric acid for phosphopeptide, HexNacHex ions from glycopeptides or linker cleavage from drug conjugate to facilitate the discovery. Once a modification is identified, the relative abundance of the modified species can be estimated from the intensity ratio of the modified vs. native peptides. It should be noted that the modification can alter the ionization efficiency of the peptide and more accurate quantitation should be obtained from synthetic peptide standards. For monoclonal antibodies, we identify and report the relative abundance for commonly occurring modifications including pyroglutamation, glycosylation, lysine deletion, deamidation and oxidation. The result is suitable for regulatory filing purpose.
Disulfide bond in protein therapeutics have an important effect on the integrity of the final product. Mapping of disulfide bonds in the proteins of interest provides insight into the integrity of a protein therapeutic and significant contribution to process efficiency.
Peptide De Novo sequencing is the analytical process that derives a peptide’s amino acid sequence from its tandem mass spectrum (MS/MS) without the assistance of a sequence database. De Novo sequencing is necessary when the protein sequence is unavailable.
Glycoprotein is first detected in intact form employing LC HR MS (high-resolution mass spectrometer), which may exhibit the confirmation of protein identity, N- and c-termini, certain post-translational modifications (PTMs) such as glycosylation status (meaning glycan heterogeneity), phosphorylation and oxidation. A more detailed understanding of glycosylation may be undertaken as peptide mapping to map glycan site and occupancy; as glycan releases by PNGase F etc, derivatized and analyzed with LC-Fluorescence, MS, and MS/MS.
Gel separated proteins can be readily identified with our nanoLC-nanospray system which provides sensitivity sufficient for silver stained gels. The gel can be an SDS-PAGE from an immunoprecipitation experiment or 2 D gel with coomassie blue or silver stain. Complex protein samples such as cell and tissue extracts can be analyzed with fractionation using either SDS-PAGE on protein level or high pH reverse phase on peptide level prior to LC-MS analysis. Thousands of proteins can be confidently identified and reported.