Differential adduction of proteins vs. deoxynucleosides by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro.
The reactions of two model mutagenic and carcinogenic alkylating agents, N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS), with proteins and deoxynucleosides in vitro, were investigated. The protein work used an approach involving trypsin digestion and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). This technique permitted identification of the specific location of protein adduction by both MNU and MMS with commercial apomyoglobin and human hemoglobin, under physiological conditions. MNU treatment resulted in predominantly carbamoylation adducts on the proteins, but in contrast only methylated protein adducts were found following treatment with MMS. Further analyses, using TurboSequest, and the Scoring Algorithm for Spectral Analysis (SALSA), revealed that MNU carbamoylation was specific for modification of either the N-terminal valine or the free amino group in lysine residues of apomyglobin and human hemoglobin. However, MMS methylation modified the N-terminal valine and histidine residues of the proteins. Despite their clear differences in protein modifications, MNU and MMS formed qualitatively the same methylated deoxynucleoside adduct profiles with all four deoxynucleosides in vitro under physiological conditions. In light of their different biological potencies, where MMS is considered a 'super clastogen' while MNU is a 'super mutagen', these differences in reaction products with proteins vs. deoxynucleosides may indicate that these two model alkylating agents work via different mechanisms to produce their mutagenic and carcinogenic effects.
Zhang
Fagen
F
Toxicology and Environmental Research & Consulting, The Dow Chemical Company, 1803 Building, Midland, MI 48674, USA. fzzhang@dow.com
Bartels
Michael J
MJ
Pottenger
Lynn H
LH
Gollapudi
B Bhaskar
BB
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003853:Deoxyribonucleosides / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D008741:Methyl Methanesulfonate / Q000737:chemistry*; D008770:Methylnitrosourea / Q000737:chemistry*; D008969:Molecular Sequence Data; D009153:Mutagens / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001059:Apoproteins; D003853:Deoxyribonucleosides; D006454:Hemoglobins; D009153:Mutagens; D009211:Myoglobin; D011506:Proteins; C010857:apomyoglobin; D008770:Methylnitrosourea; D008741:Methyl Methanesulfonate