Negative ion dissociation of peptides containing hydroxyl side chains

The dissociation of deprotonated peptides containing hydroxyl side chains was studied by electrospray ionization coupled with Fourier transform ion cyclotron resonance (ESI-FTICR) via sustained off-resonance irradiation collision induced dissociation (SORI-CID). Dissociation under post-source decay (PSD) conditions was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). This work included hexapeptides with one residue of serine, threonine, or tyrosine and five inert alanine residues. During SORI-CID and PSD, dissociation of [M-H](-) yielded c- and y-ions. Side-chain losses of formaldehyde (HCHO) from serine-containing peptides, acetaldehyde (CH(3)CHO) from threonine-containing peptides, and 4-methylene-2,5-cycohexadienone (C(7)H(6)O) from tyrosine-containing peptides were generally observed in the negative ion PSD and SORI-CID spectra. Side-chain loss occurs much less from tyrosine-containing peptides than from serine- and threonine-containing peptides. This is probably due to the bulky side chain of tyrosine, resulting in steric hindrance and poor geometry for dissociation reactions. Additionally, a selective cleavage leading to the elimination of the C-terminal residue from [M-H](-) was observed from the peptides with serine and threonine at the C-terminus. This cleavage does not occur in the dissociation of peptides with an amide group at the C-terminus or peptides with neutral or basic residues at the C-terminus. It also does not occur with tyrosine at the C-terminus. Both the C-terminal carboxylic acid group and the hydroxyl side chain of the C-terminal residue must play important roles in the mechanism of C-terminal residue loss. A mechanism involving both the C-terminal carboxylic acid group and a hydroxyl side chain of serine and threonine is proposed.     

Fragmentation characteristics of peptide-metal ion adducts under matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometric conditions

Fragmentation reactions of sodium-cationized enkephalin peptides generated by matrix-assisted laser desorption/ionization were studied using post-source decay (PSD) with a reflectron time-of-flight mass spectrometer. Several matrices and analyte-matrix sample preparation methods were evaluated for high-intensity ion currents that could last for the entire PSD analysis. A triple dried-droplet sample preparation procedure with 2,5-dihydroxybenzoic acid as the matrix was found to yield abundant longer-lasting ion signals of the peptide-Na(+) ion adducts. The principal decay product of these adduct ions is the [b(n-1) + Na + OH](+) ion, which provides an unambiguous identification of the C-terminal residue of a peptide. In some peptides, the loss of a second residue from the C-terminus is also observed. No other sequence-specific ions were observed.     

Fragmentation of the deprotonated ions of peptides containing cysteine, cysteine sulfinic acid, cysteine sulfonic acid, aspartic acid, and glutamic acid

We examined the fragmentation of the electrospray-produced [M-H]- and [M-2H]2- ions of a number of peptides containing two acidic amino acid residues, one being aspartic acid (Asp) or glutamic acid (Glu), and the other being cysteine sulfinic acid [C(SO2H)] or cysteine sulfonic acid [C(SO3H)], on an ion-trap mass spectrometer. We observed facile neutral losses of H2S and H2SO2 from the side chains of cysteine and C(SO2H), respectively, whereas the corresponding elimination of H2SO3 from the side chain of C(SO3H) was undetectable for most peptides that we investigated. In addition, the collisional activation of the [M-H]- ions of the C(SO2H)-containing peptides resulted in the cleavage of the amide bond on the C-terminal side of the C(SO2H) residue. Moreover, collisional activation of the [M-2H]2- ions of the above Asp-containing peptides led to the cleavage of the backbone N-Calpha bond of the Asp residue to give cn and/or its complementary [zn-H2O] ions. Similar cleavage also occurred for the singly deprotonated ions of the otherwise identical peptides with a C-terminal amide functionality, but not for the [M-H]- ions of same peptides with a free C-terminal carboxylic acid. Furthermore, ab initio calculation results for model cleavage reactions are consistent with the selective cleavage of the backbone N-Calpha bond in the Asp residue.     

Dissociation of multiply charged negative ions for hirudin (54–65), fibrinopeptide B, and insulin A (oxidized)

Collision-induced dissociation (CID) was performed on multiply deprotonated ions from three commercial peptides: hirudin (54-65), fibrinopeptide B, and oxidized insulin chain A. Ions were produced by electrospray ionization in a Fourier transform ion cyclotron resonance mass spectrometer. Each of these peptides contains multiple acidic residues, which makes them very difficult to ionize in the positive mode. However, the peptides deprotonate readily making negative ion studies a viable alternative. The CID spectra indicated that the likely deprotonation sites are acidic residues (aspartic, glutamic, and cysteic acids) and the C-terminus. The spectra are rife with c, y, and internal ions, although some a, b, x, and z ions form. Many of the fragment ions were formed from cleavage adjacent to acidic residues, both N- and C-terminal to the acidic site. In addition, neutral loss (e.g., NH3, CH3, H2O, and CO2) was prevalent from both the parent ions and from fragment ions. These neutral eliminations were often indicative of specific amino acid residues. The fragmentation patterns from several charge states of the parent ions, when combined, provide significant primary sequence information. These results suggest that negative mode CID of multiply deprotonated ions provides useful structural information and can be worthwhile for highly acidic peptides that do not form positive ions in abundance.     

The effects of chromium(III) coordination on the dissociation of acidic peptides

The complexes formed between chromium(III) and synthetic acidic peptides were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion-cyclotron resonance (FT-ICR) mass spectrometer equipped with electrospray ionization (ESI). Neutral peptides and peptides containing one, two, and multiple acidic residues were studied. Formation of [M + Cr-2H]+ occurred for all peptides. Three noteworthy features were found in the CID spectra of [M + Cr-2H]+. The first is that fewer fragment ions were produced from [M + Cr-2H]+ than from [M + H]+. The reason may be that multiple coordination between chromium(III) and carboxylate or carbonyl groups hinders the production of fragment ions by continuing to bind pieces of the peptide to chromium(III) after cleavage of bonds within the peptide. The second feature is loss of CO from [M + Cr-2H]+ and [y(n) + Cr-H]+. A mechanism involving coordination of chromium(III) with carboxylate groups is proposed to rationalize elimination of CO. The third feature is that chromium(III) is retained in all fragment ions, indicating strong binding of the metal ion to the peptides. The complex [M + 2Cr-5H]+ is formed as the peptide chain length and number of acidic residues increases. Longer peptides have more sites to coordinate with chromium(III) and more conformational flexibility. In addition, formation of [M + Cr-2H]+ from AGGAAAA-OCH(3), which has no carboxylic acid groups, suggests that chromium(III) can coordinate with sites on the peptide backbone, albeit in low abundance. In the negative mode, [M + Cr-4H](-) was only found for peptides containing four or more carboxylic acid groups. This is consistent with deprotonated carboxylic acid groups being involved in chromium(III) coordination and with chromium existing in the 3 + state in the gas-phase ions.     

Impact of proline and aspartic acid residues on the dissociation of intermolecularly crosslinked peptides

The dissociation of intermolecularly crosslinked peptides was evaluated for a series of peptides with proline or aspartic acid residues positioned adjacent to the crosslinking sites (lysine residues). The peptides were crosslinked with either disuccinimidyl suberate (DSS) or disuccinimidyl L-tartrate (DST), and the influence of proline and aspartic acid residues on the fragmentation patterns were investigated for precursor ions with and without a mobile proton. Collisionally activated dissociation (CAD) spectra of aspartic acid-containing crosslinked peptide ions, doubly-charged with both protons sequestered, were dominated by cleavage C-terminal to the Asp residue, similar to that of unmodified peptides. The proline-containing crosslinked peptides exhibited a high degree of internal ion formation, with the resulting product ions having an N-terminal proline residue. Upon dissociation of the doubly-charged crosslinked peptides, twenty to fifty percent of the fragment ion abundance was accounted for by multiple cleavage products. Crosslinked peptides possessing a mobile proton yielded almost a full series of b- and y-type fragment ions, with only proline-directed fragments still observed at high abundances. Interestingly, the crosslinked peptides exhibited a tendency to dissociate at the amide bond C-terminal to the crosslinked lysine residue, relative to the N-terminal side. One could envision updating computer algorithms to include these crosslinker specific product ions--particularly for precursor ions with localized protons--that provide complementary and confirmatory information, to offer more confident identification of both the crosslinked peptides and the location of the crosslink, as well as affording predictive guidelines for interpretation of the product-ion spectra of crosslinked peptides.     

Atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry of sulfonic acid derivatized tryptic peptides.

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) and ion trap mass spectrometry have been used to study the fragmentation behavior of native peptides and peptide derivatives prepared for de novo sequencing applications. Sulfonic acid derivatized peptides were observed to fragment more extensively and up to 28 times more efficiently than the corresponding native peptides. Tandem mass spectra of native peptides containing aspartic or glutamic acids are dominated by cleavage on the C-terminal side of the acidic residues. This significantly limits the amount of sequence information that can be derived from those compounds. The MS/MS spectra of native tryptic peptides containing oxidized Met residues show extensive loss of CH(3)SOH and little sequence-specific fragmentation. On the other hand, the tandem mass spectra of derivatized peptides containing Asp, Glu and oxidized Met show much more uniform fragmentation along the peptide backbone. The AP-MALDI tandem mass spectra of some derivatized peptides were shown to be qualitatively very similar to the corresponding vacuum MALDI postsource decay mass spectra, which were obtained on a reflector time-of-flight instrument. However, the ion trap mass spectrometer offers several advantages for peptide sequencing relative to current reflector time-of-flight instruments including improved product ion mass measurement accuracy, improved precursor ion selection and MS(n). These latter capabilities were demonstrated with solution digests of model proteins and with in-gel digests of 2D-gel separated proteins.     

A Systematic Study of Acidic Peptides for b-Type Sequence Scrambling

A systematic study was carried out to examine the effects of acidic amino acid residues and the position of the acidic group on the cyclization of b ions. The study utilized the model C-terminal amidated peptides XAAAAAA, AXAAAAA, AAXAAAA, AAAXAAA, AAAAXAA, AAAAAXA, AAAAAAX, XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX, where X is a glutamic acid (E) or aspartic acid (D) residue. The CID mass spectra of b _n (where n = 7 and 8) ions derived from XAAAAAA, AAAXAAA, AAAAAAX and XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX exhibited very similar fragmentation patterns for both the glutamic and the aspartic acid peptide series. The CID mass spectra of MH⁺ derived from model peptides presented substantial direct and non-direct sequence b ions. The results indicate that b ions produced from acidic peptides can also undergo head-to-tail cyclization, which is the reason for the formation of the non-direct sequence b ions. The b ion spectra derived from the peptides became more complex as the number of acidic residues in the peptides increased. Side chains of glutamic and aspartic acid did not inhibit the cyclization of the b ions. Substantial water elimination was observed in all CID spectra of b ₇ and b ₈ ions. Finally, the preferential cleavage of glutamic or aspartic acid residues from macrocyclic structures of b ions was also investigated under various collision energy conditions.     

Novel rearranged ions observed for

C-terminal rearrangement ions [b(n-1) + H2O] (where n refers to the total number of residues of peptides) are frequently observed for peptides which contain basic amino acid(s), especially arginine, at or near their N termini in low- and high-energy collision-induced dissociation or post-source decay (PSD) spectra. Here we report a novel rearrangement, associated with PSD for serine- or threonine-containing peptides that are susceptible to C-terminal rearrangement. Based on PSD analyses of serine- or threonine-containing bradykinin and its analogs, which have been ethyl-esterified or 18O labeled at their C termini, the [b(k) + H2O] (where k denotes the position adjacent to the left of the Ser/Thr residue) ion is generally thought to be formed by the transfer of the hydroxyl moiety of a serine or threonine residue to the carbonyl group of the residue to its left accompanied by the loss of the remaining C-terminal portion of the peptide. When the Ser/Thr is at or near the C terminus, the present [b(k) + H2O] ion could be formed via two pathways, i.e., the Ser/Thr-related rearrangement and the conventional C-terminal rearrangement, which has been clearly verified by 18O labeling at the C terminus. In addition, the ions which are formally designated as [y(m)b(l) + H2O], where y(m)b(l) denotes a b-type internal ion, are also briefly described.     

Further studies on the fragmentation of protonated ions of peptides containing aspartic acid, glutamic acid, cysteine sulfinic acid, and cysteine sulfonic acid

Here we examined the fragmentation, on a quadrupole ion-trap mass spectrometer, of the protonated ions of a group of peptides containing one arginine and two different acidic amino acids, one being aspartic acid (Asp) or glutamic acid (Glu) and the other being cysteine sulfinic acid [C(SO2H)] or cysteine sulfonic acid [C(SO3H)]. Our results showed that, upon collisional activation, the cleavage of the peptide bond C-terminal to C(SO2H) is much more facile than that of the peptide bond C-terminal to Asp, Glu, or C(SO3H). There is no significant difference, however, in susceptibility to cleavage of peptide bonds that are C-terminal to Asp, Glu, and C(SO3H). To understand these experimental observations, we carried out B3LYP/6-31G* density functional theory calculations for a model cleavage reaction of GXG --> b2 + Gly, in which X is Asp, Glu, C(SO2H), or C(SO3H). Our calculation results showed that the cleavage reaction is thermodynamically more favorable when X = C(SO2H) than when X = Asp or C(SO3H). We attributed the less facile cleavage of the amide bond after Glu to that the formation of a six-membered ring b ion for Glu-bearing peptides is kinetically not as favorable as the formation of a five-membered ring b ion for peptides containing the other three acidic amino acids. The results from this study may provide useful tools for peptide sequencing.     

Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses. Copyright © 2010 John Wiley & Sons, Ltd.     

Electron transfer dissociation with supplemental activation to differentiate aspartic and isoaspartic residues in doubly charged peptide cations

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z ^• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.     

Studies on the dephosphorylation of phosphotyrosine-containing peptides during post-source decay in matrix-assisted laser desorption/ionization

Phosphorylation of tyrosine residues in proteins is a common regulatory mechanism, although it accounts for less than 1% of the total O-phosphate content in proteins. Whereas aromatic phosphorylation sites can be identified by a number of different analytical techniques, sequence analysis of phosphotyrosine-containing proteins at the low picomole or even femtomole level is still a challenging task. This paper describes the post-source decay in matrix-assisted laser desorption/ionization mass spectrometry of phosphotyrosine-containing model peptides by comparing their fragmentation behavior with sequence-homologous unphosphorylated peptides. Whereas the parent ions showed significant losses of HPO3, all phosphorylated fragment ions of the b- and y-series displayed only minor dephosphorylated signals, which often were not detectable. Surprisingly, one of the studied phosphotyrosine-containing sequences displayed, in addition to the [M + H - 80]+ ion, a more abundant [M + H - 98]+ ion, which could be explained by elimination of phosphoric acid. This dephosphorylation pattern was very similar to the patterns obtained for phosphoserine- and phosphothreonine-containing peptides. Because the dephosphorylation pattern of the parent ion is often used to identify modified amino acids in peptides, we investigated possible dephosphorylation mechanisms in detail. Therefore, we substituted single trifunctional amino acid residues and incorporated deuterated phosphotyrosine residues. After excluding direct elimination of phosphoric acid from tyrosine, we could show that the obtained loss of H3PO4 depends on aspartic acid and arginine residues. Most likely the HPO3 group is transferred to aspartic acid followed by cleavage of phosphoric acid forming a succinimide. On the other hand, arginine appears to induce the H3PO4 loss by protonation of phosphotyrosine leaving a phenyl cation.     

Characteristics of photodissociation at 193 nm of singly protonated peptides generated by matrix-assisted laser desorption ionization (MALDI)

Photodissociation (PD) at 193 nm of various singly protonated peptides was investigated. These include peptides with an arginine residue at the C-terminus, N-terminus, at both termini, inside the chain, and those without an arginine residue. Monoisotopomeric selection was made for the precursor ions. Interference from the post-source decay (PSD) product signals was reduced as much as possible by using the deflection system (reported previously) and subtracting the remaining signals from the laser-on signals. The presence of an arginine residue and its position inside the peptide were found to significantly affect the PD spectra, as reported previously. Presence of a proline, aspartic acid, or glutamic acid residue hardly affected the PD spectral patterns. By comparing the PD spectra obtained at a few different wavelengths, it is concluded that the dissociation of the photoexcited ions occurs in their ground electronic states. Tentative explanations for the observed spectral correlations based on the statistical picture for the reactions are also presented.     

天冬氨酸作为非特异性吸附抑制剂在二氧化钛选择性富集磷酸肽中的应用

二氧化钛富集法作为目前使用最为广泛的金属氧化物富集磷酸肽的方法,在富集过程中常常对富含天冬氨酸和谷氨酸的酸性非磷酸化肽段存在一定的非特异性吸附作用。这些肽段与磷酸化肽段一同被富集,降低了磷酸肽富集的选择性。传统方法中使用的非特异性吸附抑制剂常会对质谱的电喷雾离子源造成污染,因而限制了其在液相色谱-质谱联用(LC-MS)系统中的应用。本研究将天冬氨酸作为一种新型的非特异性吸附抑制剂加入到二氧化钛富集体系中,并分别对3种和9种标准蛋白质酶切肽段混合物进行富集实验,同时与添加另一种非特异性吸附抑制剂——谷氨酸以及不添加任何非特异性吸附抑制剂的富集体系进行了富集效果的比较。结果表明,天冬氨酸可以有效地提高二氧化钛对磷酸肽富集的选择性。将添加天冬氨酸的二氧化钛富集体系应用于鼠肝全蛋白质磷酸肽的富集中,同样取得了很好的效果,表明天冬氨酸在复杂的生物样本的磷酸肽富集中也同样具有良好的应用前景。此外,由于天冬氨酸在反相色谱中极易被洗脱去除,从而避免了传统抑制剂对LC-MS系统离子源的污染问题。     

Neutral loss of amino acid residues from protonated peptides in collision-induced dissociation generates N- or C-terminal sequence ladders

The widespread occurrence of the neutral loss of one to six amino acid residues as neutral fragments from doubly protonated tryptic peptides is documented for 23 peptides with individual sequences. Neutral loss of amino acids from the N-terminus of doubly charged tryptic peptides results in doubly charged y-ions, forming a ladder-like series with the ions [M + 2H](2+) = y(max) (2+), y(max - 1) (2+), y(max - 2) (2+), etc. An internal residue such as histidine, proline, lysine or arginine appears to favor this type of fragmentation, although it was sometimes also observed for peptides without this structure. For doubly protonated non-tryptic peptides with one of these residues at or near the N-terminus, we observed neutral loss from the C-terminus, resulting in a doubly charged b-type ion ladder. The analyses were performed by Q-TOF tandem mass spectrometry, facilitating the recognition of neutral loss ladders by their 2+ charge state and the conversion of the observed mass differences into reliable sequence information. It is shown that the neutral loss of amino acid residues requires low collision offset values, a simple mechanistic explanation based on established fragmentation rules is proposed and the utility of this neutral loss fragmentation pathway as an additional source for dependable peptide sequence information is documented.     

Fragmentation reactions of protonated peptides containing glutamine or glutamic acid

A variety of protonated dipeptides and tripeptides containing glutamic acid or glutamine were prepared by electrospray ionization or by fast atom bombardment ionization and their fragmentation pathways elucidated using metastable ion studies, energy-resolved mass spectrometry and triple-stage mass spectrometry (MS(3)) experiments. Additional mechanistic information was obtained by exchanging the labile hydrogens for deuterium. Protonated H-Gln-Gly-OH fragments by loss of NH(3) and loss of H(2)O in metastable ion fragmentation; under collision-induced dissociation (CID) conditions loss of H-Gly-OH + CO from the [MH - NH(3)](+) ion forms the base peak C(4)H(6)NO(+) (m/z 84). Protonated dipeptides with an alpha-linkage, H-Glu-Xxx-OH, are characterized by elimination of H(2)O and by elimination of H-Xxx-OH plus CO to form the glutamic acid immonium ion of m/z 102. By contrast, protonated dipeptides with a gamma-linkage, H-Glu(Xxx-OH)-OH, do not show elimination of H(2)O or formation of m/z 102 but rather show elimination of NH(3), particularly in metastable ion fragmentation, and elimination of H-Xxx-OH to form m/z 130. Both the alpha- and gamma-dipeptides show formation of [H-Xxx-OH]H(+), with this reaction channel increasing in importance as the proton affinity (PA) of H-Xxx-OH increases. The characteristic loss of H(2)O and formation of m/z 102 are observed for the protonated alpha-tripeptide H-Glu-Gly-Phe-OH whereas the protonated gamma-tripeptide H-Glu(Gly-Gly-OH)-OH shows loss of NH(3) and formation of m/z 130 as observed for dipeptides with the gamma-linkage. Both tripeptides show abundant formation of the y(2)' ion under CID conditions, presumably because a stable anhydride neutral structure can be formed. Under metastable ion conditions protonated dipeptides of structure H-Xxx-Glu-OH show abundant elimination of H(2)O whereas those of structure H-Xxx-Gln-OH show abundant elimination of NH(3). The importance of these reaction channels is much reduced under CID conditions, the major fragmentation mode being cleavage of the amide bond to form either the a(1) ion or the y(1)' ion. Particularly when Xxx = Gly, under CID conditions the initial loss of NH(3) from the glutamine containing dipeptide is followed by elimination of a second NH(3) while the initial loss of H(2)O from the glutamic acid dipeptide is followed by elimination of NH(3). Isotopic labelling shows that predominantly labile hydrogens are lost in both steps. Although both [H-Gly-Glu-Gly-OH]H(+) and [H-Gly-Gln-Gly-OH]H(+) fragment mainly to form b(2) and a(2) ions, the latter also shows elimination of NH(3) plus a glycine residue and formation of protonated glycinamide. Isotopic labelling shows extensive mixing of labile and carbon-bonded hydrogens in the formation of protonated glycinamide.     

Mass spectrometric differentiation of alpha- and beta-aspartic acid in a pseudo-tetrapeptide thrombosis inhibitor and its isomer

The pseudo-tetrapeptide designated here as RGD (N-ethyl-N-[1-oxo-4-(4-piperidinyl) butyl] glycyl-L-alpha- aspartyl-3-cyclohexyl-L-alaninamide) and its isomer with beta-aspartic acid rather than alpha-aspartic acid were examined using electrospray ionization (ESI) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). RGD has potential as a thrombosis inhibitor and the isomer, designated here as isopeptide, is an inactive instability product; hence, means were sought to distinguish the two. Both isomers give a protonated parent on ESI and fragments typical of peptides on sustained off resonance irradiation collision-induced decomposition (SORI-CID). Cleavage at the aspartic acid (b(3)) is the dominant process in both isomers, although a significant b(2) and smaller a(2)" and c(2)" peaks are also observed. More distinctive are peaks observed at b(3)-H(2)O, b(3)-(CO + CO(2)) and, only in the case of the RGD, b(3) - (H(2)O + CO). SORI CID on the b(3) ion indicates that, of these distinctive peaks, only the b(3)-(CO + CO(2)) comes from decomposition of the b(3) ion. On this basis, a mechanism is suggested for b(3) formation, involving proton transfer from a back-bone carbonyl to the aspartic acid side-chain carboxyl group. Such an intramolecular proton transfer involves rings of different sizes for the two isomers, providing a basis for the different SORI energy dependences. A mechanism suggested for the formation of the b(3)-H(2)O fragments also involves proton transfer to the aspartic acid side chain carboxyl group. This leads to concomitant H(2)O loss and amide bond cleavage, giving the b(3)-H(2)O ions with ketene moieties resulting from the water loss. According to the suggested mechanism, the observed loss of CO (verified by SORI-CID on the b(3)- H(2)O ion) from the RGD b(3)-H(2)O peak results in a secondary carbocation stabilized by an adjacent nitrogen. The unobserved loss of CO from the b(3)-H(2)O ion, formed by the suggested mechanism from the isopeptide, would give an unstable primary carbocation lacking a neighboring nitrogen. The mechanism, thus, only rationalizes the observation of a b(3)-(H(2)O + CO) fragment in RGD and not in the isopeptide. The isomers can be distinguished on the basis of this unique peak or on the basis of the different SORI energy dependence of the formation of the b(3) ions.     

酶促合成肽的研究 II: 含羧基侧链氨基酸的肽的酶促合成

用嗜热菌酶酶促合成了一系列含门冬氨酸、谷氨酸的肽. 实验证明, 缩合反应中羧基组份中的门冬氨酸β-羧基或谷氨酸γ-羧基毋需保护. 同时也研究了邻近氨基酸残基对缩合反应的影响.     

Sequencing of peptides phosphorylated on serines and threonines by post-source decay in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In the era of complete genome sequences, biochemical and medical research will focus more on the dynamic proteome of a cell. Regulation of proteins by post-translational modifications, which are not determined by the gene sequence, are already intensively studied. One example is phosphorylation of serines and threonines, probably the single most common cellular regulatory mechanism. In this paper we describe the sequencing of mono- and bisphosphorylated peptides, including identification of the phosphorylation sites, by post-source decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition to dephosphorylation of the parent ions, we studied the influence of the phosphate group on the fragmentation of peptides. Generally, peptides phosphorylated on serine and threonine residues displayed no difference in their fragmentation patterns. The intensities of the resulting fragment ion signals depend only on the peptide sequence and not on either the phosphorylated amino acid or its position in the peptide chain. Phosphorylation increased the bond cleavage C-terminal to the phosphorylation site more than 10-fold, resulting in abundant signals, which typically dominated the PSD spectra. The produced C-terminally phosphorylated b-type fragment ions showed characteristic dephosphorylated fragment ions b(n) -H(3)PO(4) (-98 Da) and b(n) -HPO(3) (-80 Da) of higher abundances than the phosphorylated fragment ion. As a second layer to identify the phosphorylation site, all internally phosphorylated fragment ions were accompanied by minor, but always detectable, signals of the dephosphorylated fragment ions. Interpretation of PSD spectra of phosphopeptides was not more complicated than for unphosphorylated peptides, despite the increased number of obtained fragment ion signals.