Characterization of Nα-Fmoc-protected ureidopeptides by electrospray ionization tandem mass spectrometry (ESI-MS/MS): differentiation of positional isomers.
Four pairs of positional isomers of ureidopeptides, FmocNH-CH(R(1))-ϕ(NH-CO-NH)-CH(R(2))-OY and FmocNH-CH(R(2))-ϕ(NH-CO-NH)-CH(R(1))-OY (Fmoc = [(9-fluorenyl methyl)oxy]carbonyl; R(1) = H, alkyl; R(2) = alkyl, H and Y = CH(3)/H), have been characterized and differentiated by both positive and negative ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). The major fragmentation noticed in MS/MS of all these compounds is due to --N--CH(R)--N--bond cleavage to form the characteristic N- and C-terminus fragment ions. The protonated ureidopeptide acids derived from glycine at the N-terminus form protonated (9H-fluoren-9-yl)methyl carbamate ion at m/z 240 which is absent for the corresponding esters. Another interesting fragmentation noticed in ureidopeptides derived from glycine at the N-terminus is an unusual loss of 61 units from an intermediate fragment ion FmocNH = CH(2) (+) (m/z 252). A mechanism involving an ion-neutral complex and a direct loss of NH(3) and CO(2) is proposed for this process. Whereas ureidopeptides derived from alanine, leucine and phenylalanine at the N-terminus eliminate CO(2) followed by corresponding imine to form (9H-fluoren-9-yl)methyl cation (C(14)H(11) (+)) from FmocNH = CHR(+). In addition, characteristic immonium ions are also observed. The deprotonated ureidopeptide acids dissociate differently from the protonated ureidopeptides. The [M - H](-) ions of ureidopeptide acids undergo a McLafferty-type rearrangement followed by the loss of CO(2) to form an abundant [M - H - Fmoc + H](-) which is absent for protonated ureidopeptides. Thus, the present study provides information on mass spectral characterization of ureidopeptides and distinguishes the positional isomers.
National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad 500 607, Andhra Pradesh, India.
M
M
Ramesh
B
B
Raju
R
R
Srinivas
V V
VV
Sureshbabu
N
N
Narendra
B
B
Vasantha
D000596:Amino Acids / Q000737:chemistry*; D005449:Fluorenes / Q000737:chemistry*; D007536:Isomerism; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014508:Urea / Q000737:chemistry*
D000596:Amino Acids; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010455:Peptides; D014508:Urea