Protein Analyses

Intact Protein Analysis by LC-MS at Rilas

At Rilas, we perform intact protein analysis using Liquid Chromatography-Mass Spectrometry (LC -MS) with a Quadrupole Time-of-Flight (QToF) mass spectrometer that offers unparalleled precision and insight into protein structure, composition, and purity. This cutting-edge technique enables a comprehensive understanding of proteins in their intact form, revealing valuable data essential for research, drug development, and biopharmaceutical production.

Exact Mass Determination

QToF LC-MS provides highly accurate mass measurements, allowing for precise determination of the exact molecular weight of intact proteins. By measuring the mass-to-charge ratio (m/z) of the entire protein molecule, this technique delivers exact mass values with sub-ppm (parts per million) accuracy, which is crucial for identifying and confirming protein identity and post translational modifications (PTMs). The ability to resolve closely related protein isoforms or variants ensures confidence in characterization and quality control, including in our purification services.

Glycan analysis on intact NIST mAb (RM 8671), showing the 5 major, commonly observed glycans for monoclonal antibodies (combinations of G0F, G1F, and G2F). Smaller identified peaks include GlcNac hydrolysis of those major glycoforms.

Glycosylation Analysis

Glycosylation is a critical post translational modification that can significantly affect protein function, stability, and immunogenicity. Rilas is capable of identifying and quantifying glycosylation patterns, including the attachment sites, glycan composition, and heterogeneity of glycosylation structures. This is specially valuable in the analysis of therapeutic proteins, such as monoclonal antibodies (se Antibody/ADCs page for more), where glycosylation plays a vital role in efficacy and safety. The high-resolution and separation capabilities of Rilas’ unique HRIM LCMS makes it possible to resolve different glycoform isomers and evaluate the influence of glycosylation on protein behavior and activity with unparalleled precision

HILIC-MS based acquisition of N-linked glycans released from a glycoprotein batch through enzymatic treatment. Identified glycans were matched to database entries through several metrics including retention time, mass, UV fluorescence, and fragmentation MS spectrum. Released glycan assays like this can be performed on single proteins for batch analysis or on protein mixtures to ascertain more global glycomics information.

Structural Characterization

Understanding a protein’s structural features is essential for drug design and functional analysis. Rilas provides insights into the overall architecture of intact proteins, enabling the identification of disulfide bond linkages, non-covalent and covalent interactions, and conformational states. By analyzing the intact protein in its native state, researchers can observe structural integrity, protein folding, and potential unfolding or aggregation under varying conditions, which is critical for biopharmaceutical stability studies. This helps in assessing how well a protein retains its functional form under different formulations or environmental conditions, including in our purification pipeline.

Purity Analysis

LCUV-MS is a powerful tool for assessing the purity of protein samples. By resolving intact proteins based on their molecular mass, this method allows for the detection of impurities, truncations, degradation products, and isoforms that might not be apparent with traditional methods. The ability to quantify the relative abundance of the protein of interest versus impurities enables precise purity assessment, which is vital in biopharmaceutical manufacturing and quality control. The sensitivity and resolution of MS can detect even low levels of contaminants, ensuring that the final product meets required purity standards.

Sample Identification

LC-MS provides an exceptional platform for identifying unknown proteins or confirming known proteins in complex mixtures. The high-resolution mass spectra generated from intact proteins can be matched against databases to pinpoint protein identities based on their exact mass. Additionally, when coupled with chromatography, it offers a clear separation of individual protein species in a sample, allowing for comprehensive proteomic profiling and sample fingerprinting. This capability is particularly valuable in proteomics, where comprehensive identification and characterization of proteins from biological samples are essential.

Why Choose Rilas for Intact Protein Analysis?

The HRIM-QToF mass spectrometer offers a unique combination of high resolution, high sensitivity, isomeric separation, and fast data acquisition, making it ideal for intact protein analysis. With its ability to provide detailed information on molecular weight, glycosylation, structural features, purity, and identification, LC-MS at Rilas is a versatile and reliable tool for any biopharmaceutical application. Whether you're developing new therapeutics, conducting basic research, or ensuring the quality of your product, Rilas will provide you with the actionable insights you need to drive success.

Instrumentation:
Agilent 6545XT AdvanceBio LC/Q-TOF
Agilent 1260 Infinity II Bio-inert
Agilent 1290 Infinity II HPLC/MS