Following incubation, samples were dialyzed overnight at 4C against two changes of the phosphate buffer (2L each), and then applied to separate aminophenylboronate columns (1.5×10 cm) previously equilibrated with 50mM phosphate buffer, Mogroside IVe pH 7.4. Treatment of galactated HSA with sodium borohydride stabilized the condensed sugars on the protein and yielded discrete fragmentation patterns by tandem MS, allowing reliable identification of HSAs galactation sites. LC/ESI/MS, in combination with tandem MS, revealed the principal sites of galactation in HSA were the amino groups of lysine 12, 233, 281/276, 414 and 525. Lysyl residues 12, 233, 276, and 525 were previously reported as privileged sites for the nonenzymatic binding of D-glucose with HSA. Keywords:Nonenzymatic glycation, Galactated HSA, Amadori adducts, Mass spectrometry, MALDI-TOF/MS, Tandem MS, Peptide mapping == Introduction == The nonenzymatic glucation of human serum albumin (HSA) and human hemoglobin has been extensively studied and has been the subject of several reviews [15]. What has made the glucation of these proteins clinically significant is usually that they both provide for an assessment of glycemic control in diabetes, with glucated HSA and glucated hemoglobin serving as sensitive indicators of short term and long term hyperglycemic control, respectively [68]. Studies in our laboratory and those of others have exhibited that thein vivoglycation of HSA and hemoglobin does not occur with D-glucose only and that in certain conditions such as galactosemia, the high levels of D-galactose in blood can also promote the Mogroside IVe galactation of HSA and hemoglobin [913]. The glucation and galactation of proteins begins with the formation of an unstable Schiff base that occurs between the carbonyl group of the reducing sugars D-glucose or D-galactose and the free amino groups on proteins. This Schiff base then rearranges to form a Mogroside IVe more stable Amadori product that with time forms a heterogeneous group of compounds referred to Advanced Glycation End Products (AGEs) [1416]. The mechanistic similarity in the formation of nonenzymatically glucated and galactated proteins [14,15] suggests that akin to diabetes with glucated proteins, the monitoring of galactated proteins may provide a valuable tool for the management of patients with classical galactosemia, a rare genetic disorder characterized with increased D-galactose and galactitol levels in tissues and body fluids [9,1720]. To explore this possibility and develop an assay specific for galactated proteins, we focused our attention to HSA and to its amino acid sites of galactation. Emphasis was placed on the vulnerable sites of galactation for two reasons: 1) to design genuine galactated peptides similar to those found in HSA for developing monoclonal antibodies to the protein, and 2) to determine if the sites of galactation coincided with HSAs reported glucation sites. Mass spectrometry using ESI and MALDI-MS has been successfully applied to the study of protein glucation in diabetes, particularly, when mapping for glycated peptides, characterizing advanced glucation end products and determining the number of D-glucose residues condensed with proteins [21,22]. This paper describes the application of a sodium borohydride dependent mass peptide mapping method for identifying HSAs galactation sites. In this procedure, in vitro prepared galactated HSA was first reduced with sodium borohydride, and the resulting protein was then digested with trypsin. The tryptic digests were analyzed by LC/ESI/MS and tandem MS which yielded quality spectra with minimal neutral water losses. Neutral water loss behavior is usually a phenomenon commonly observed in collision induced dissociation (CID) of glycated peptide ions. During CID, the preferential release of water from the labile Mogroside IVe Amadori adducts results in poor production of sequence specific ions from the peptide backbone. Consequently, this yields a CID spectrum with little or no useful information Fgf2 impeding the identification of peptide sequences and sites of glycation [2325]. The reduction of galactated HSA with sodium borohydride appeared to not only stabilize the linkages between the nonenzymatically reacted sugars and the peptides, but also yielded discrete fragmentation patterns by tandem MS allowing for the easy and reliable identification of HSAs galactation sites. In this report, we describe the galactation sites in HSA and compare our findings with previously reported glucation sites for the protein. == Materials and Methods == == Reagents == Sterile filtered normal human serum screened and tested for and found non-reactive for Hepatitis B & C and non-reactive for Human Immunodeficiency Virus (HIV) antibody was purchased from the BioBank of Sera Care Life Sciences, Inc. (Oceanside, CA). D-galactose, aminophenylboronic acid resins, silica C18resins, solvents and trypsin for HPLC had been purchased from Sigma Aldrich Chemical substance Co. (St. Louis, MO). AffiGel Blue, Bio-Gel-P-150, regular chromatographic Coomassie and resins G 250 reagent had been from Bio-Rad lnc. (Richmond, CA). Unless indicated otherwise, all the reagents and products had been from Pierce Chemical substance Business (Rockford, IL). == Evaluation of Protein Focus == Protein amounts had been dependant on Coomassie G 250 reagent using human being albumin as the typical proteins. == Purification of Albumin from Human being Serum == HSA.