title: Identification and quantitative studies of protein immobilization sites by stable isotope labeling and mass spectrometry.
abstract: A method was developed for characterizing immobilization sites on a protein based on stable isotope labeling and MALDI-TOF mass spectrometry. The model for this work was human serum albumin (HSA) immobilized onto silica by the Schiff base method. The immobilized HSA was digested by various proteolytic enzymes in the presence of normal water, while soluble HSA was digested in (18)O-enriched water for use as an internal standard. These two digests were mixed and analyzed, with the (18)O/(16)O ratio for each detected peptide then being measured. Several peptides in the tryptic, Lys-C, and Glu-C digests gave significantly higher (18)O/(16)O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA (e.g., K4, K41, K190, K225, K313, and K317). It was also possible from these results to quantitatively rank these sites in terms of the relative degree to which each might take part in immobilization. This method is not limited to HSA and silica but can be used with other proteins and supports.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
authors.forename: Chunling
authors.initials: C
authors.lastname: Wa
authors.forename: Ron L
authors.initials: RL
authors.lastname: Cerny
authors.forename: David S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D000595:Amino Acid Sequence; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D012709:Serum Albumin; D018698:Glutamic Acid; D008239:Lysine