Enhancement of charge remote fragmentation in protonated peptides by high-energy CID MALDI-TOF-MS using “cold” matrices

116

181

One Hundred Fifty-Seven nm photodissociation of singly protonated peptides generates unusual distributions of fragment ions. When the charge is localized at the C-terminus of the peptide, spectra are dominated by x-, v-, and w-type fragments. When it is sequestered at the N-terminus, a-and d-type ions are overwhelmingly abundant. Evidence is presented suggesting that the fragmentation occurs via photolytic radical cleavage of the peptide backbone at the bond between the ␣-and carbonyl-carbons followed by radical elimination to form the observed daughter ions. Low-energy fragmentation appears to be well described by the mobile proton model according to which vibrational excitation of the analyte leads to charge mobility [12]. Transfer of the charge proton to either the backbone carbonyl oxygen or amide nitrogen enables charge-induced cleavage of the peptide backbone. This process yields primarily b-and y-type fragment ions according to the standard nomenclature shown below [13,14].Higher energy activation methods involving collisions with gas molecules or surfaces can enable chargeremote fragmentation [15][16][17]. However, even in these cases, apparently, protonated peptides still generate some fragments through charge-directed processes [17]. Immobilization of the charge, either by using metal adducts [18] or charged chemical modifications [19 -22], has been used to inhibit charge-directed fragmentation. In these cases, primarily a-, d-, and w-type fragments are observed. Several mechanisms have been proposed for charge-remote peptide fragmentation, some involving homolytic radical cleavage [14,23].Electron capture dissociation (ECD) [24] and the recently reported electron-transfer dissociation (ETD)[25] appear to be nonthermal processes. These phenomena generate c-and z-type fragment ions upon the addition of an electron to a multiply charged protein or peptide ion. Two mechanisms have been proposed, one involving reactions of a free hydrogen atom generated when an electron is captured by a charged site on the analyte ion [26], and the other involving localization of the ϳ6 eV of energy that is generated upon charge neutralization. This energy then induces fragmentation through an excited electronic state [27]. Implicit in this second mechanism is the suggestion that techniques which excite appropriate electronic states can lead to unique, nonergodic fragmentation even for molecules as large as peptides and proteins [24,27]. This can occur if ions reach a dissociative electronic state and fragment before the energy is redistributed throughout the molecule. Electronic to vibrational relaxation, resulting in nonspecific excitation is an important competing process that often inhibits nonergodic processes, and rates of relaxation are predicted to increase with the size of the molecule [28].Lasers are powerful tools that can also be used to excite molecules to specific energy levels with either multiphoton or single photon processes. Light is not affected by the electric or magnetic fields of mass spectrometers, s...

Section: X-type Ionssupporting

confidence: 83%

Section: Secondary Fragmentationmentioning

confidence: 99%

Pathways of Peptide Ion Fragmentation Induced by Vacuum Ultraviolet Light

Cui

Thompson

Reilly

2005

116

181

“…In this study, both CHCA and 2,5-DHB were chosen for investigation of the "direct analysis" phenomenon. 2,5-DHB is another MALDI matrix compound, which has been reported to represent a more effective matrix for use in studies of labile phospho-and glycopeptides due to its comparatively "cool" nature (i.e., reduced energy transfer to analyte during the desorption/ionization process) [12][13][14]. The relatively "hot" nature of CHCA, which under laser desorption conditions causes increased metastable decay of phosphopeptide ions, reduces ion signal intensity for intact phosphopeptide detection, as one might expect.…”

Section: Discussionmentioning

confidence: 99%

Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS

Hart

Waterfield

Burlingame

et al. 2002

We have revisited the direct analysis experiments reported by Tomer and co-workers in the MALDI-TOFMS analysis of phosphopeptide-loaded immobilized metal ion affinity chromatography (IMAC) beads (Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2000, 11, 273-282). The results described herein provide no evidence to support a laser-induced direct desorption of phosphopeptides chelated on IMAC beads. However, we have established that solubilization of mono-phosphopeptides from their immobilized Fe 3ϩ -NTA chelates does occur effectively in solutions containing certain MALDI matrices. Particularly effective is 2,5-dihydroxybenzoic acid (2,5-DHB), which apparently forms a stronger chelation complex with Fe 3ϩ -NTA than mono-phosphopeptides. With regard to the disparity observed between the low pH value of MALDI matrices (saturated 2,5-DHB aq ϳ pH 2) and the high pH values of conventional IMAC eluents (typically above pH 7), we have also investigated the influence of eluent pH on the recovery of phosphopeptides from IMAC media. Finally, we have confirmed the importance of employing ammonium dihydrogen phosphate as buffer to achieve effective liberation of mono-and all poly-phosphopeptide species from I mmobilized metal ion affinity chromatography (IMAC) has been used for a number of years for the selective enrichment of phosphopeptides from proteolytic digest mixtures containing both phosphorylated and non-phosphorylated components [1][2][3][4][5][6][7][8]. The established methods have largely relied upon the use of high-pH elution buffers to disrupt the interaction of phosphopeptides remaining bound to the metal ioncontaining IMAC media after separation of all other peptides [2][3][4][5].In addition to the usual conditions documented above for solubilization of peptides bound in their immobilized, chelated form attached to the resin, evidence has been presented which suggests that monophosphopeptides may be released directly from the resin during the MALDI process [7,9]. It was recognized that the ferric IMAC resin must bind multiple phosphorylated components as well, but that laser irradiation does not easily dissociate these components from the agarose IMAC beads [7].While it was suggested that laser desorption directly from IMAC beads occurs particularly for mono-phosphorylated peptide components, in these reports it was also questioned whether or not the IMAC analytes are still bound to the beads through their affinity interaction immediately prior to laser desorption [9,10]. Therefore, the possibility exists that "elution" from the beads into the MALDI matrix solution/crystals has in fact taken place prior to MALDI desorption and mass analysis.Of course, there would be clear advantages in having a protocol that permits the direct analysis of phosphopeptides from IMAC beads, particularly if the analyte species thus detected could be qualitatively and quantitatively representative of the components bound to the resin. However, if phosphopeptide desorption takes place directly fro...

“…For example, matrix materials with acid (e.g., 2,5-dihydroxybenzoic acid) or hydroxy groups (e.g., dithranol) compete with the FA for Li ϩ and because of the high ratio of matrix to analyte, FA lithium adduction is minimized. However, highly electron-deficient matrix materials (e.g., TCNQ) readily donate Li harge remote fragmentation (CRF) has been shown by Gross and others to have considerable analytical utility because fragmentation remote to the charge site occurs without carbon skeletal rearrangements [1][2][3][4][5][6][7][8][9]. Thus, features such as double-bond and branching positions of various functional groups can be determined directly by mass spectrometry (MS).…”

mentioning

confidence: 99%

Charge-remote fragmentation of lithiated fatty acids on a TOF-TOF instrument using matrix-ionization

Trimpin

Clemmer

McEwen

2007

In numerous studies charge remote fragmentation (CRF) has been shown to be a powerful technique for determination of primary structure by allowing location of double bonds, various functional groups, and branching in a variety of compound types directly by mass spectrometry. Instrumentation and ionization methods traditionally used for CRF, however, are becoming rare, in large part because ESI and MALDI have to a significant extent replaced them. Here we demonstrate that by selecting a matrix that promotes rather than suppresses ionization of fatty acids (FA) by lithium ion adduction, and using a TOF-TOF mass spectrometer for high-energy collisional activation, CRF ions are produced that allow location of double-bond and branching positions. Further, we show that by using solvent-free MALDI sample preparation methods, thus eliminating the inherent segregation of the hydrophobic fatty acid from the hydrophilic LiCl that can occur during the evaporation of solvent, the desired [FAϪHϩ2Li] ϩ ions are greatly enhanced. Because FAs can be vaporized using laser desorption, matrix assistance in desorption of the fatty acid may occur, but is not necessary. However, the matrix plays a crucial role in enhancing or suppressing ionization. For example, matrix materials with acid (e.g., 2,5-dihydroxybenzoic acid) or hydroxy groups (e.g., dithranol) compete with the FA for Li ϩ and because of the high ratio of matrix to analyte, FA lithium adduction is minimized. However, highly electron-deficient matrix materials (e.g., TCNQ) readily donate Li ϩ to FAs because of the instability associated with being positively charged. (J Am Soc Mass