Purification and characterization of the central segment of prothymosin-alpha: methodology for handling highly acidic peptides.
Prothymosin-alpha is a highly acidic protein consisting of 110 amino acids. The central segment of this protein, residues 51-89, is thought to be involved in metal binding which may be necessary for its physiological function. To carry out studies of this peptide, this central segment was synthesized in a linear fashion using Fmoc-based methods on rink amide MBHA resin. However, this peptide could not be purified with the typical straightforward approach of RP HPLC followed by negative mode electrospray ionization mass spectrometry (ESI-MS). This was attributed to the high proportion of acidic residues: 26 out of the 39 residues are aspartic and glutamic acids. The acidity of the peptide prevented retention on the RP HPLC column. Additionally, the ability of the highly negatively charged peptide to retain sodium ions prevented molecular weight determination with ESI-MS. A systematic approach to the purification of this highly acidic peptide was undertaken. Ultimately, strong anion exchange chromatography was used to purify the peptide. Extensive desalting using dialysis was required prior to ESI-MS, and the choice of the buffer proved to be critical. In the end, a purification method was devised that yielded a highly purified peptide and is readily compatible with analysis by ESI-MS.
Wilson
Christopher L
CL
Department of Chemistry, East Carolina University, East 5th Street, Greenville, NC 27858, USA.
Monteith
William B
WB
Danell
Allison S
AS
Burns
Colin S
CS
D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003956:Dialysis / Q000379:methods; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry* / Q000302:isolation & purification*; D012964:Sodium / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013947:Thymosin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification
D011498:Protein Precursors; C040782:prothymosin alpha; D013947:Thymosin; D012964:Sodium