PMC

CEX-HPLC profile of the mAb. Inset – iso-Asp content of the unfractionated mAb and its charge isoforms [].

Generic workflow of mass spectrometric identification and quantification of isomerized residues.

Top panel: extracted ion chromatogram for isomerized peptide L(D->isoD)LAGR. Bottom panel: ECD MS/MS spectra for LisoDLAGR (upper), and spectra for LDLAGR (lower). *D indicates the presence of iso-Asp [].

Spontaneous deamidation of aspargine (L-Asn), isomerization of aspartic acid (L-Asp) residues and their repair by the PIMT enzyme using S-adenosylmethionine (SAM) as the methyl donor. L-Succinimide reversely racemizes to D-succinimide, which leads to a variety of isomerized products. The nonenzymatic reactions denoted by the thick arrows are faster than those denoted by the thin arrows. SAH represents S-adenosylhomocysteine.

Tandem mass spectra derived by collision-induced dissociation (CID) of the (M + 2H)²⁺ precursor ions of the peptide VVSVLTVVHQDWLNGK (a) and its three isoforms: succinimide (b), iso-Asp (c), and Asp (d) []. The two last isoforms were assigns based on the HPLC retention times (), as the CID MS/MS spectra of these molecules are very similar.

ESI spectra of 13+ molecular ions of RNase: (A) untreated and (B–E) after deamidation that caused a mass increase of 1.0, 1.8, 3.7, and 4.4 Da, respectively. (A) Best fits of the theoretical abundance distribution corresponding to the protein deamidated at n and n + 1 sites, respectively. (B–E) Best fits of the indicated fractional number of deamidations [].

Base peak ion chromatograms of (M + 2H)²⁺ precursor ions of the four different isoforms of the peptide VVSVLTVVHQDWLNGK incubated at pH 7.5 and 37 °C in 100 mM Tris buffer for different periods of time: normal (N), succinimide (Su), iso-Asp (iso-D) and L-Asp (D) [].

Identification of deamidated Asn residues in the tetanus toxin C fragment (TTCF) protein. Deamidation position in the sample TTCF37 compared to the control TTCF4 was performed by MALDI MS upon protein digestion with trypsin and tryptic peptides separation by reversed phase HPLC. The peptides 1214–1223 (DGNAFNNLDR) and 1179–1191 (YTPNNEIDSFVK) that showed positive molecular mass shift compared to the control were sequenced by tandem MS analysis to determine the sites of deamidation, Asn residues 1219 and 1183 [].

A comparison of protein staining and 3H-methylation patterns indicates a wide range of isoaspartate content in proteins from the PIMT knock-out mouse. A - Coomassie Blue stain of a PVDF membrane after ³H-methylation and fluorography. B - ³H fluorogram of the same membrane prior to staining. Protein spots that were subsequently identified by peptide-mass fingerprinting are marked. Rectangles mark proteins that appear to have a relatively high level of isoaspartate per unit protein; diamonds and circles mark proteins with intermediate and low levels of isoaspartate, respectively [].