Bioconjugation of ribonuclease A: a detailed chromatographic and mass spectrometric analysis of chemical modification by a cross-linking reagent.
The modification of ribonuclease A with the heterobifunctional cross-linker, 4-succinimdyloxycarbonyl-methyl-alpha-[2-pyridyldithio]-toluene (SMPT) is described. RNase A has 11 potential sites of modification by the SMPT reagent. Tracking the two-dimensional separation and proteolytic digestion of SMPT-modified RNase A with ESI/FTICR-MS and HPLC/ESI/QIT-MS demonstrates the detailed information about number of SMPT modifications and sites of modification that can be obtained by application of these techniques. Analysis of native and modified RNase A tryptic digests by ESI/FTICR-MS resulted in the identification of the sites of modification. Semiquantitative results of the reactivity of certain lysine residues toward the coupling reagent SMPT are presented. Two sites (lysines 1 and 37) are highly reactive, while three sites (lysines 41, 61, and 104) appear to be unreactive toward SMPT under the conditions used. Experimental results demonstrate that quantitative comparison of relative intensities of peptide sequences of different charge states is not possible. No correlation was found between number of basic residues and sensitivity to detection. Digestion of the modified and unmodified RNase A by subtilisin followed by examination by HPLC/ESI/QIT-MS and MS(n) enabled further investigation of modification on lysines 1 and 7, including modification at the epsilon- and alpha-amino positions on lysine 1.
Méndez
T J
TJ
Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611, USA.
Johnson
J V
JV
Nichols
L S
LS
Lang
G H
GH
Eyler
J R
JR
Powell
D H
DH
Richardson
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D020860:Subtilisin / Q000378:metabolism; D013388:Succinimides / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D010455:Peptides; D013388:Succinimides; C058151:4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene; D012259:Ribonuclease, Pancreatic; D014357:Trypsin; D020860:Subtilisin; D008239:Lysine