Mass spectrometric approaches to study protein structure and interactions in lyophilized powders.
Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (N fast, N slow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.
Department of Industrial and Physical Pharmacy, Purdue University.
Balakrishnan S
BS
Moorthy
Department of Industrial and Physical Pharmacy, Purdue University.
Lavanya K
LK
Iyer
Department of Industrial and Physical Pharmacy, Purdue University.
Elizabeth M
EM
Topp
D000577:Amides / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000379:methods*; D005612:Freeze Drying / Q000379:methods; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*
D000577:Amides; D010455:Peptides; D011506:Proteins