193 nm photodissociation of larger multiply-charged biomolecules

A tandem time-of-flight mass spectrometer for the study of photodissociation of biopolymer ions generated by matrix-assisted laser desorption ionization was designed and constructed. A reflectron with linear and quadratic (LPQ) potential components was used. Characteristics of the LPQ reflectron and its utility as the second stage analyzer of the tandem mass spectrometer were investigated. Performance of the instrument was tested by observing photodissociation of [M ϩ H] ϩ from angiotensin II, a prototype polypeptide. Quality of the photodissociation tandem mass spectrum was almost comparable to that of the post-source decay spectrum. Monoisotopic selection of the parent ion was possible, which was achieved through the ion beam-laser beam synchronization. General theoretical considerations needed for a successful photodissociation of large biopolymer ions are also presented. [3] has revolutionized the application of mass spectrometry for the determination of molecular weights of biopolymers. The natural next step in this field is the tandem mass spectrometry, which detects fragmentation of a mass-selected parent ion. When the internal energy of a polyatomic ion acquired at the time of its formation is sufficient for its dissociation after exiting the source, it may dissociate unimolecularly during its flight to the detector, which is called the metastable ion decomposition (MID) [4]. A more popular way to supply additional energy is to introduce collision gas on the ion flight path such that some of parent ion translational energy is converted to its internal energy. This is called the collision-induced dissociation (CID) or collisionally activated dissociation (CAD) [2]. In the case of the tandem time-of-flight (TOF) mass spectrometry of ions generated by MALDI without the collision gas, the term post source decay (PSD) [5,6] rather than MID has been popular because CID may also contribute to the observed fragment ion signals. When tandem mass spectra generated by PSD are either very weak or do not contain sufficient structural information, CID [7][8][9] may be attempted by introducing collision gas intentionally. Excitation via multiple collisions is thought to be important in the CID tandem mass spectrometry of high mass biopolymers with many degrees of freedom. Recently, dissociation of multiply protonated molecules induced by electron capture, or electron capture dissociation (ECD) [10,11], is attracting a lot of attention as a method to obtain site-specific information.Photodissociation (PD) has been utilized in the field of tandem mass spectrometry mostly as a method to study the structure and dissociation dynamics of small polyatomic ions [12]. Infrared multiphoton dissociation (IRMPD) [13,14] has been used to induce dissociation of biopolymer ions also, even though a large amount of internal energy needed for such a dissociation necessitates absorption of a large number of infrared photons. When electronic transitions of chromophores by ultraviolet radiation are utilized, the number of photons that m...

mentioning

confidence: 99%

Tandem time-of-flight mass spectrometer for photodissociation of biopolymer ions generated by matrix-assisted laser desorption ionization (MALDI-TOF-PD-TOF) using a linear-plus-quadratic potential reflectron

Moon

Kim

2004

“…ECD typically causes extensive fragmentation of backbone amine bonds to produce c and z⅐ fragment ions, in contrast to the b and y fragment ions produced from the amide bond cleavage by conventional energetic fragmentation methods, such as collisionally activated dissociation (CAD) [9 -14], surface-induced dissociation (SID) [15], infrared multiphoton dissociation (IRMPD) [16], blackbody infrared dissociation (BIRD) [17,18]. Although c and z⅐ ions were initially observed in the 193 nm ultraviolet photodissociation (UVPD) mass spectra of multiply charged protein ions [19], they were the products of unexpected ECD processes: The low-energy electrons were ejected from metal surfaces by 193 nm photons (6.4 eV), and were captured by the protein ions trapped in a FTICR cell [1]. In addition, ECD is a nonergodic process (i.e., cleavage happens prior to energy randomization); the ECD backbone cleavages impart very little internal energy into the fragments, as a result, the labile side-chain modifications can be retained on the backbone fragment ions.…”

mentioning

confidence: 99%

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation

Guan

2002

Self Cite

Electron capture dissociation (ECD) has been demonstrated to be an effective fragmentation technique for characterizing the site and structure of the fatty acid modification in ghrelin, a 28-residue growth-hormone-releasing peptide that has an unusual ester-linked n-octanoyl (C8:0) modification at Ser-3. ECD cleaves 21 of 23 possible backbone amine bonds, with the product ions (c and z⅐ ions) covering a greater amino acid sequence than those obtained by collisionally activated dissociation (CAD). Consistent with the ECD nonergodic mechanism, the ester-linked octanoyl group is retained on all backbone cleavage product ions, allowing for direct localization of this labile modification. In addition, ECD also induces the ester bond cleavage to cause the loss of octanoic acid from the ghrelin molecular ion; the elimination process is initiated by the capture of an electron at the protonated ester group, which is followed by the radical-site-initiated reaction known as ␣-cleavage. emerged as a promising new tool for tandem mass spectrometry (MS/MS) of multiply charged (protonated) proteins and peptides generated by electrospray ionization (ESI) [6] using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry [7,8]. ECD typically causes extensive fragmentation of backbone amine bonds to produce c and z⅐ fragment ions, in contrast to the b and y fragment ions produced from the amide bond cleavage by conventional energetic fragmentation methods, such as collisionally activated dissociation (CAD) [9 -14], surface-induced dissociation (SID) [15], infrared multiphoton dissociation (IRMPD) [16], blackbody infrared dissociation (BIRD) [17,18]. Although c and z⅐ ions were initially observed in the 193 nm ultraviolet photodissociation (UVPD) mass spectra of multiply charged protein ions [19], they were the products of unexpected ECD processes: The low-energy electrons were ejected from metal surfaces by 193 nm photons (6.4 eV), and were captured by the protein ions trapped in a FTICR cell [1]. In addition, ECD is a nonergodic process (i.e., cleavage happens prior to energy randomization); the ECD backbone cleavages impart very little internal energy into the fragments, as a result, the labile side-chain modifications can be retained on the backbone fragment ions. This is in sharp contrast to the conventional ergodic fragmentation methods that typically eject the labile modifications before cleaving the backbone bonds. These unique attributes of ECD in providing extensive, nonergodic peptide backbone fragmentation have been demonstrated to be advantageous for localizing labile posttranslational modifications, such as ␥-carboxylation [20], sulfonation [20], glycosylation [21][22][23], and phosphorylation [24,25].In this study we examine ECD of ghrelin, a posttranslationally acylated peptide hormone that has an unusual C8:0 acylation (octanoic acid modification) at Ser-3 residue [26,27]. The ester-linked C8:0 fatty acyl moiety is essential for the activities of ghrelin, which include growth hormone secretion, feeding ...

“…MS/MS spectra of multiply charged ubiquitin ions have been collected by a variety of activation methods, including SORI-CAD [36], laserinduced dissociation [37,38], BIRD [39], ECD [20], post ion-ion reaction [40], and ETD [21]. Fragmentation of singly charged ubiquitin was also examined in a MALDI TOF-TOF instrument [7,8].…”

Section: Ubiquitinmentioning

confidence: 99%

Fragmentation of multiply-charged intact protein ions using MALDI TOF-TOF mass spectrometry

Liu

Schey

2008

Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach. ragmentation of intact protein gas-phase ions, termed top down proteomics, is becoming more frequently utilized primarily in ion trapping instruments. As was first reported by Loo et al.[1], multiply-charged intact protein ions are formed via electrospray ionization (ESI) and can be collisionally activated to produce sequence-specific fragment ions. Typically, the fragmentation of highly multiply charged intact proteins results in a product ion spectrum displaying a multitude of product ions possessing a range of charge states making spectral interpretation challenging. The high resolving power and mass accuracy of Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry has made this instrument platform the method of choice for the interpretation of the complicated product ion spectra produced by fragmentation of multiply charged protein ions. Indeed, automated interpretation algorithms have been constructed based on this approach [2,3]. Using FTMS, proteins over 200 kDa have been successfully fragmented and sequence-specific product ion assignments have been made [4]. Lower mass resolving quadrupole ion trapping instruments have also been successfully utilized for top down proteomics experiments. To meet the challenge of spectral interpretation ion/ion reactions have been employed to reduce the product ion charge by subjecting the product ion population to proton transfer reactions, thereby resulting in the formation of predominantly singly charged product ions and a more readily interpretable product ion spectrum [5]. This strategy removes the product ion charge-state ambiguities arising from dissociation of large multiply charged protein ions and has been successfully demonstrated on ions as large as elastase (25.9 kDa) although it is limited by the m/z range of the instrument [6]. Matrix-assisted laser desorption ionization tandem time-of-flight (MALDI TOF-TOF) mass spectrometry has also been used to produce product i...