Supplemental Data
Electron Induced Dissociation of Singly Deprotonated Peptides
Anastasia Kalli,$Gabriela Grigorean,‡ and Kristina Håkansson*
Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109-1055, USA
$ Current address: Proteome Exploration Laboratory, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA
‡ Current address: MS Proteomics @ The European Institute of Oncology, via Adamello 16, 20139Milan, Italy
Table S1A. Product ions observed following EID of singly deprotonated substance P-NH2. Loss of the entire side chains of phenylalanine (PhCH2, 91.055 Da) and methionine (CH2CH2SMe, 75.027 Da) were only detected following EID (see also Table S1B).
Table S1B. Product ions observed following CAD of singly deprotonated Substance P-NH2.
Table S2A. Product ions observed following EID of singly deprotonated Substance P-OH. Side chain losses from methionine (MeSMe, 62.019 Da), phenylalanine (91.055 Da), arginine (CH2CH2CH2NHC(NH)NH2, (100. 087 Da), leucine (CH2CH(CH3)CH3, 57.070 Da), and glutamine (CH2CH2CONH2, 72.045 Da) were observed in EID, but wereabsent in CAD (see also Table S2B).
Table S2B. Product ions observed following CAD of singly deprotonated Substance P-OH.
Table S3A. Product ions observed following EID of singly deprotonated LHRH-NH2.
Table S3B. Product ions observed following CAD of singly deprotonated LHRH-NH2. A loss of NH=C=NH (42.022 Da) from arginine and a loss of HCHO (30.011 Da) from serine is observed in CAD. The loss of NH=C=NH from arginine has been previously reported for dipeptides in CAD of singly deprotonated peptides formed by FAB[1]. This loss, NH=C=NH, was absent in EID (Table S3A). Product ions corresponding to multiple neutral losses are formed. For example, combined loss of the side chains of tryptophan and serine from the precursor ions is observed (the corresponding product ion is indicated as m3/4(W)/(S)). These multiple neutral losses were absent in the EID spectrum (Table S3A). The b8product ion was detected as b8-30 (HCHO) - 42 (NH=C=NH).
Table S4A. Product ions observed following EID of singly deprotonated LHRH-OH. Loss of the entire tryptophan side chain is observed in both EID and CAD (Table S4B), similar to the amidated form (Tables S3A and S3B). Characteristic side chain loss from serine was also detected. A loss of 59.038 Da is detected from the y7 product ion. This loss can be attributed to the arginine side chain (C1H5N3, 59.048 Da) and it has been previously reported in ECD [2].
Table S4B. Product ions observed following CAD of singly deprotonated LHRH-OH. Characteristic side chain loss from serine is observed. Loss of NH=C=NH from the side chain of arginine is particularly prevalent in CAD. For the amidated form of LHRH, the NH=C=NH loss was significantly less pronounced in CAD (Table S3B) and it was absent in EID (Table S3A). Similar to CAD of LHRH-NH2 (Table S3B), the b8product ion was detected as b8–30 (HCHO) – 42 (NH=C=NH).
Table S5A. Product ions observed following EID of singly deprotonated neuromedin B.
The precursor ions and the majority of product ions exhibit abundant neutral loss of CH3CHO (44.026 Da) from the threonine residue. Based on their masses, product ions at m/z = 1012.441, 898.4065, and 785.3240 can be assigned as either y-type product ions exhibiting NH3 loss, and a 44 Da loss from threonine (C2H4O1, 44.026 Da), or as z-type product ions exhibiting 45 Da loss from Thr (C2H5O1, 45.034 Da).
Table S5B. Product ions observed following CAD of singly deprotonated neuromedin B.
Similar to EID (Table S5A) the precursor ions and the majority of product ions exhibit abundant neutral loss of CH3CHO (44.026 Da) from the threonine residue. Based on its mass, the product ion at m/z = 1012.441 can be assigned as either a y-type fragment exhibiting NH3 loss, and a 44 Da loss from threonine (C2H4O1, 44.026 Da), or as a z-type fragment exhibiting 45 Da loss from Thr (C2H5O1, 45.034 Da).
Table S6A. Product ions observed following EID of singly deprotonated pEVNFSPGWGT-NH2. Characteristic CH3CHO (44.026 Da) loss was highly abundant in both EID and CAD (Table S6B). Loss of the phenylalanine side chain (91.055 Da) is observed from the y8 product ion. This [y8 – 91 (F)] product ion is also observed in CAD (Table S6B). For this peptide, the 44 Da loss could also be assigned as the loss of CONH2 (44.014 Da) from the amidated C-terminus. However, the mass error was higher for this assignment and, also, because CH3CHO loss from the Thr residue is known to be prevalent in negative ion mode MS/MS of Thr containing peptides this assignment seems more likely.
Table S6B. Product ions observed following CAD of singly deprotonated pEVNFSPGWGT-NH2. The 44 Da loss can be assigned to loss of the threonine side chain (CH3CHO, 44.026 Da), or loss of the amidated C-terminus (CONH2, 44.014 Da).However, for the [m5 – 44] product ion the assignment [m5 – 44 (44.014 Da, CONH2)] had an error higher than 15 ppm and it was therefore excluded.
Table S7A. Product ions observed following EID of singly deprotonated pEQWFWWM-NH2. Trp side chain loss (130 Da) from the precursor ions is present, similar to the peptides LHRH (Tables S3A. S4A) and neuromedin B (S5A). The c5 product ion exhibited a loss of 129 Da, instead of 130 Da, from the Trp side chain.
Table S7B. Product ions observed following CAD of singly deprotonated pEQWFWWM-NH2. Neutral loss of 45.013 Da is observed from the precursor ions. This loss corresponds to loss of HCONH2 (45.021 Da) from a glutamine residue and it has previously been observed in ECD [2], but it has not been reported in negative ion mode CAD. Neutral losses of 75.027 Da and 62.013 Da correspond to losses of CH2CH2SMe (75.027 Da) and MeSMe (62.019 Da), respectively, from the methionine side chain. Such cleavages have been previously reported in negative ion mode FAB-CAD[3]. The neutral loss of 72.045 corresponds to loss of CH2CH2CONH2from glutamine. Loss of the Trp side chain (129 Da) is particularly abundant from both precursor and product ions. One product ion, z6, exhibited a loss of 130 Da instead of 129 Da from the Trp side chain.
Table S8A. Product ions observed following EID of singly deprotonated bradykinin. Similar to the peptides LHRH-NH2 (Table S3A), LHRH-OH (Table S4A), and pEVNFSPGWGT-NH2 (Table S6A), neutral loss of HCHO from Ser is observed. Side chain losses from phenylalanine (91.055 Da) and arginine (100.087 Da) are also observed. These side chain losses were absent in CAD of bradykinin (Table S8B), although Phe side chain loss was observed in CAD of the peptide pEVNFSPGWGT-NH2 (Table S6B). Neutral loss of NH=C=NH (42 Da) from arginine is also present, although this loss is more dominant in CAD (Table S8B).
Table S8B. Product ions observed following CAD of singly deprotonated bradykinin. Similar to the peptides LHRH-NH2 (Table S3B), LHRH-OH (Table S4B), and pEVNFSPGWGT-NH2 (Table S6B), neutral loss of HCHO from Ser is observed. Neutral loss of NH=C=NH (42 Da) from arginine is also present.
Table S9A. Product ions observed following EID of singly deprotonated H-WHWLQL-OH. A loss of 129 Da from a tryptophan side chain was detected from the product ions z5 and b3 similar to the peptide pEQWFWWM-NH2 (Table S7A).
Table S9B. Product ions observed following CAD of singly deprotonated H-WHWLQL-OH. A loss of 129 Da from a tryptophan side chain is detected from the product ions c3 and c4.
Table S10A. Product ions observed following EID of singly deprotonated cholecystokinin. Side chain loss from methionine (62.019 Da) is observed. This loss was absent in CAD (Table S10B). The product ion at m/z = 764.2586 can be assigned as either [b6 -H2O]-,[c6 - H2O - NH3]-, or as [a7 - 107 (Y)]-.
Table S10B. Product ions observed following CAD of singly deprotonated cholecystokinin. The product ion at m/z = 764.2561 can be assigned as either [b6 -H2O]-, [c6 - H2O - NH3]- or as [a7 - 107 (Y)]-.
Table S11A. Product ions observed following EID of singly deprotonated CH3CO-RRA(pS)VA-OH.
Table S11B. Product ions observed following CAD of singly deprotonated CH3CO-RRA(pS)VA-OH.
Table S12A. Product ions observed following EID of singly deprotonated tyrosine 2-sulfated cholecystokinin. Neutral losses of 59.013 Da, 75.027 Da, and 130.066 Da are observed from the precursor ions. These losses can be attributed to the loss of CH2COOH (59.013 Da) from the aspartic acid, loss of CH2CH2SMe (75.027 Da) from the Met residue, and loss of C9H8N (130.066 Da) from the Trp residue. These losses were absent following CAD (Table S12B). The product ion at m/z = 781.2811 can be assigned as either [y2 -2H]-, or [c6 - SO3 - H2O]- and the product ion at m/z = 764.2509 can be assigned as [b6 - SO3 - H2O]-, or as [c6 - SO3 - H2O - NH3]- .
Table S12B. Product ions observed following CAD of singly deprotonated tyrosine 2-sulfated cholecystokinin. The product ion at m/z = 781.2820 can be assigned as either [y2 -2H]-, or as [c6 - SO3 - H2O]- and the product ion at m/z = 764.2511 can be assigned as [b6 - SO3 - H2O]-,or as [c6 - SO3 - H2O - NH3]-
References.
1.Waugh, R. J.; Bowie, J. H.; Gross, M. L. Collision-Induced Dissociations of Deprotonated Peptides - Dipeptides Containing Asn, Arg and Lys. Aust. J. Chem.1993,46, 693-702.
2.Cooper, H. J.; Hudgins, R. R.; Hakansson, K.; Marshall, A. G. Characterization of amino acid side chain losses in electron capture dissociation. J. Am. Soc. Mass Spectrom.2002,13, 241-249.
3.Waugh, R. J.; Bowie, J. H.; Gross, M. L. Collision-Induced Dissociations of Deprotonated Peptides - Dipeptides Containing Methionine or Cysteine. Rapid Commun. Mass Spectrom.1993,7, 623-625.
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