共查询到20条相似文献,搜索用时 31 毫秒
1.
Håkansson K Cooper HJ Emmett MR Costello CE Marshall AG Nilsson CL 《Analytical chemistry》2001,73(18):4530-4536
Glycoproteins are a functionally important class of biomolecules for which structural elucidation presents a challenge. Fragmentation of N-glycosylated peptides, employing collisionally activated dissociation, typically yields product ions that result from dissociation at glycosidic bonds, with little occurrence of dissociation at peptide backbone sites. We have applied two dissociation techniques, electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD), in a 7-T Fourier transform ion cyclotron resonance mass spectrometer, in the investigation of an N-glycosylated peptide from an unfractionated tryptic digest of the lectin of the coral tree, Erythrina corallodendron. ECD provided c and z. ions derived from the peptide backbone, with no observed loss of sugars. Cleavage at 11 of 15 backbone amine bonds was observed. The lack of cleavage at sites located close to the glycosylated asparagine residue may result from steric blocking by the glycan. IRMPD provided abundant fragment ions, primarily through dissociation at glycosidic linkages. The monosaccharide composition and the presence of three glycan branch sites could be determined from the IRMPD fragments. The two types of spectra, obtained with the same instrument, thus provide complementary structural information about the glycopeptide. The current result extends the applicability of ECD for glycopeptide analysis to N-glycosylated peptides and to peptides containing branched, highly substituted glycans. 相似文献
2.
Håkansson K Chalmers MJ Quinn JP McFarland MA Hendrickson CL Marshall AG 《Analytical chemistry》2003,75(13):3256-3262
We have mounted a permanent on-axis dispenser cathode electron source inside the magnet bore of a 9.4-T Fourier transform ion cyclotron resonance mass spectrometer. This configuration allows electron capture dissociation (ECD) to be performed reliably on a millisecond time scale. We have also implemented an off-axis laser geometry that enables simultaneous access to ECD and infrared multiphoton dissociation (IRMPD). Optimum performance of both fragmentation techniques is maintained. The analytical utility of performing either ECD or IRMPD on a given precursor ion population is demonstrated by structural characterization of several posttranslationally modified peptides: IRMPD of phosphorylated peptides results in few backbone (b- and y-type) cleavages, and product ion spectra are dominated by neutral loss of H3PO4. In contrast, ECD provides significantly more backbone (c- and z*-type) cleavages without loss of H3PO4. For N-glycosylated tryptic peptides, IRMPD causes extensive cleavage of the glycosidic bonds, providing structural information about the glycans. ECD cleaves all backbone bonds (except the N-terminal side of proline) in a 3-kDa glycopeptide with no saccharide loss. However, only a charge-reduced radical species and some side chain losses are observed following ECD of a 5-kDa glycopeptide from the same protein. An MS3 experiment involving IR laser irradiation of the charge-reduced species formed by electron capture results in extensive dissociation into c- and z-type fragment ions. Mass-selective external ion accumulation is essential for the structural characterization of these low-abundance (modified) peptides. 相似文献
3.
Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry 总被引:12,自引:0,他引:12
Of methods for dissociation of multiply charged peptide and protein ions, electron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational modifications as glycosylation and carboxylation. Here this capability is successfully extended to phosphorylation, for which collisionally activated dissociation (CAD) can cause extensive loss of H3PO4 and HPO3. As shown here, these losses are minimal in ECD spectra, an advantage for measuring the degree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra give complementary backbone cleavages for identifying modification sites. For a 24-kDa heterogeneous phosphoprotein, bovine beta-casein, activated ion ECD cleaved 87 of 208 backbone bonds that identified a phosphorylation site at Ser-15, and localized three more among Ser-17,-18, -19, and -22 and Thr-24, and the last among four other sites. This is the first direct site-specific characterization of this key post-translational modification on a protein without its prior degradation, such as proteolysis. 相似文献
4.
Leymarie N Berg EA McComb ME O'Connor PB Grogan J Oppenheim FG Costello CE 《Analytical chemistry》2002,74(16):4124-4132
Proline-rich proteins (PRPs), including collagens, complement 1q, and salivary PRPs, are unusually difficult to sequence by mass spectrometry, due to the high efficiency of cleavage at the amide bond on the N-terminal of proline residues and the consequently low relative abundance of fragment arising from cleavages at other amide bonds. To fully characterize these proteins by mass spectrometry, specialized approaches to fragmentation are needed for the peptides with high proline content. Our work reported herein focused on the analysis of the set of peptides derived by tryptic cleavage of the salivary protein PRP-3. Two methods of fragmentation were compared: Collision-induced dissociation (CID) and electron capture dissociation (ECD). The data obtained demonstrated that ECD spectra of peptides containing more than 30% proline residues are simpler and easier to interpret than are CID spectra of those peptides. Factors that limit the two methods of fragmentation include the complexity of information contained in the CID spectra and the low efficiency of ECD processes. A complementary approach using both decomposition methods provides more complete and interpretable sequence information and yielded >93% sequence coverage for the 11-kDa PRP-3 isolated from human saliva. 相似文献
5.
Statistical characterization of ion trap tandem mass spectra from doubly charged tryptic peptides 总被引:7,自引:0,他引:7
Collision-induced dissociation (CID) is a common ion activation technique used to energize mass-selected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra. 相似文献
6.
Activated ion electron capture dissociation for mass spectral sequencing of larger (42 kDa) proteins 总被引:3,自引:0,他引:3
In previous studies, electron capture dissociation (ECD) has been successful only with ionized smaller proteins, cleaving between 33 of the 153 amino acid pairs of a 17 kDa protein. This has been increased to 99 cleavages by colliding the ions with a background gas while subjecting them to electron capture. Presumably this ion activation breaks intramolecular noncovalent bonds of the ion's secondary and tertiary structure that otherwise prevent separation of the products from the nonergodic ECD cleavage of a backbone covalent bond. In comparison to collisionally activated dissociation, this "activated ion" (AI) ECD provides more extensive, and complementary, sequence information. AI ECD effected cleavage of 116, 60, and 47, respectively, backbone bonds in 29, 30, and 42 kDa proteins to provide extensive contiguous sequence information on both termini; AI conditions are being sought to denature the center portion of these large ions. This accurate "sequence tag" information could potentially identify individual proteins in mixtures at far lower sample levels than methods requiring prior proteolysis. 相似文献
7.
Electron capture dissociation (ECD) is a promising method for de novo sequencing proteins and peptides and for locating the positions of labile posttranslational modifications and binding sites of noncovalently bound species. We report the ECD of a synthetic peptide containing 10 alanine residues and 6 lysine residues uniformly distributed across the sequence. ECD of the (M + 2H)(2+) produces a limited range of c (c(7)-c(15)) and z (z(9)-z(15)) fragment ions, but ECD of higher charge states produces a wider range of c (c(2)-c(15)) and z (z(2)-z(6), z(9)-z(15)) ions. Fragmentation efficiency increases with increasing precursor charge state, and efficiencies up to 88% are achieved. Heating the (M + 2H)(2+) to 150 degrees C does not increase the observed range of ECD fragment ions, indicating that the limited products are due to backbone cleavages occurring near charges and not due to effects of tertiary structure. ECD of the (M + 2Li)(2+) and (M + 2Cs)(2+) produces di- and monometalated analogues of the same c and z ions observed from the (M + 2H)(2+), with the abundance of dimetalated fragment ions increasing with fragment ion mass, a result consistent with the metal cations being located near the peptide termini to minimize Coulombic repulsion. In stark contrast to the ECD results, collisional activation of cesiated dications overwhelmingly results in ejection of Cs(+). The abundance of cesiated fragment ions formed from ECD of the (M + Cs + Li)(2+) exceeds that of lithiated fragment ions by 10:1. ECD of the (M + H + Li)(2+) results in exclusively lithiated c and z ions, indicating an overwhelming preference for neutralization and cleavage at protonated sites over metalated sites. These results are consistent with preferential neutralization of the cation with the highest recombination energy. 相似文献
8.
The feasibility of obtaining the collision-induced dissociation (CID) spectra of multiply charged peptide ions produced by electrospray ionization in a simple and inexpensive single-quadrupole mass spectrometer is demonstrated. Collisional activation was carried out in the high-pressure region between the capillary exit and the skimmer entrance to the mass analyzer. The CID of multiply charged peptide ions is very efficient, and the observed fragment ion intensities are typically 1-5% of the parent ion intensity prior to CID. About 70 pmol of the peptide is consumed in obtaining each CID spectrum. Spectra obtained by CID of multiply charged ions from bradykinin, angiotensin II, two peptides with features similar to tryptic peptides, and a synthetic analogue of a component of TGF-alpha containing two disulfide bonds are shown. The influence of the primary structure of the peptide on the observed fragmentation pathways is discussed. Although the present single-quadrupole configuration is simple and effective, the inability to choose a particular parent ion for collisional activation makes it less powerful than the triple-quadrupole configuration for mixtures of peptides and peptide samples that yield more than one charge state in the normal mass spectrum. However, it has the potential for inexpensively obtaining sequence information of proteins at high sensitivity by analyzing the pure tryptic peptides obtained by on-line or off-line chromatographic separation of tryptic digests. 相似文献
9.
For the backbone dissociation of large (29 kDa) multiply charged protein ions in the gas phase by electron capture, the main experimental challenges are juxtaposition of the electron and ion for efficient capture, dissociation of tertiary noncovalent bonds that prevent product separation, and minimization of secondary electron capture that destroys larger product ions. A simple alternative methodology is described in which electrons (0.03-100 microA, 0.1-15 eV) are first impinged on a gas pulse in the ion cell of a Fourier transform mass spectrometer, followed by ion beam introduction. For carbonic anhydrase, the resulting plasma conditions produce 87% efficiency for electron capture; a single spectrum yields 512 product ions of 237 different masses from cleavage of 183 of the 258 interresidue bonds, while two spectra cleave 197 of these bonds. The problem of secondary dissociation of product ions is reduced by plasma conditions in which product ions are formed near electrons whose velocities are unfavorable and whose capture cross sections no longer have a square dependence on charge. One plasma ECD spectrum of ubiquitin provides its sequence de novo except for two residue pairs. ECD of casein identifies 126 of 208 interresidue cleavages, providing direct and specific characterization of all its 26 Ser/Thr/Tyr phosphorylation sites. 相似文献
10.
Kleinnijenhuis AJ Duursma MC Breukink E Heeren RM Heck AJ 《Analytical chemistry》2003,75(13):3219-3225
Electron capture induced dissociation (ECD) and collisionally activated dissociation (CAD) experiments were performed on four lanthionine bridge-containing antibiotics. ECD of lantibiotics produced mainly c and z* ions, as has been observed previously with other peptides, but more interestingly, the less common c* and z ions were observed in abundance in the ECD spectra. These fragments specifically resulted from the cleavage of both a backbone amine bond and the thioether bond in a lanthionine bridge. ECD seemed to induce mainly cleavages near the lanthionine bridges. This fragmentation pattern indicates that lanthionine bridges play a key role in the selectivity of the ECD process. A new mechanism is postulated describing the formation of c* and z ions. Comparative low-energy CAD did not show such specificity. Nondissociative ECD products were quite abundant, suggesting that relatively stable double and triple radicals can be formed in the ECD process. Our results suggest that ECD can be used as a tool to identify the C-terminal attachment site of lanthionine bridges in newly discovered lantibiotics. 相似文献
11.
Fast mapping of disulfide bonds in proteins containing multiple cysteine residues is often required in order to assess the integrity of the tertiary structure of proteins prone to degradation and misfolding or to detect distinct intermediate states generated in the course of oxidative folding. A new method of rapid detection and identification of disulfide-linked peptides in complex proteolytic mixtures utilizes the tendency of collision-activated peptide ions to lose preferentially side chains of select amino acids in the negative ion mode. Cleavages of cysteine side chains result in efficient dissociation of disulfide bonds and produce characteristic signatures in the fragment ion mass spectra. While cleavages of other side chains result in insignificant loss of mass and full retention of the peptide ion charge, dissociation of external disulfide bonds results in physical separation of two peptides and, therefore, significant changes of both mass and charge of fragment ions relative to the precursor ion. This feature allows the fragment ions generated by dissociation of external disulfide bonds to be easily detected and identified even if multiple precursor ions are activated simultaneously. Such broadband selection of precursor ions for consecutive activation is achieved by lowering the dc/rf amplitude ratio in the first quadrupole filter of a hybrid quadrupole time-of-flight mass spectrometer. The feasibility of the new method is demonstrated by partial mapping of disulfide bridges within a 37-kDa protein containing 16 cysteine residues and complete disulfide mapping within a lysozyme (14.5 kDa) containing 8 cysteine residues. In addition to detecting peptide pairs connected by a single external disulfide, the new method is also shown to be capable of identifying peptides containing both external and internal disulfide bonds. The two major factors determining the efficiency of disulfide mapping using the new methodology are the effectiveness of proteolysis and the ability of the resulting proteolytic fragments to form multiply charged negative ions. 相似文献
12.
Electron capture dissociation for structural characterization of multiply charged protein cations 总被引:15,自引:0,他引:15
Zubarev RA Horn DM Fridriksson EK Kelleher NL Kruger NA Lewis MA Carpenter BK McLafferty FW 《Analytical chemistry》2000,72(3):563-573
For proteins of < 20 kDa, this new radical site dissociation method cleaves different and many more backbone bonds than the conventional MS/MS methods (e.g., collisionally activated dissociation, CAD) that add energy directly to the even-electron ions. A minimum kinetic energy difference between the electron and ion maximizes capture; a 1 eV difference reduces capture by 10(3). Thus, in an FTMS ion cell with added electron trapping electrodes, capture appears to be achieved best at the boundary between the potential wells that trap the electrons and ions, now providing 80 +/- 15% precursor ion conversion efficiency. Capture cross section is dependent on the ionic charge squared (z2), minimizing the secondary dissociation of lower charge fragment ions. Electron capture is postulated to occur initially at a protonated site to release an energetic (approximately 6 eV) H. atom that is captured at a high-affinity site such as -S-S- or backbone amide to cause nonergodic (before energy randomization) dissociation. Cleavages between every pair of amino acids in mellitin (2.8 kDa) and ubiquitin (8.6 kDa) are represented in their ECD and CAD spectra, providing complete data for their de novo sequencing. Because posttranslational modifications such as carboxylation, glycosylation, and sulfation are less easily lost in ECD than in CAD, ECD assignments of their sequence positions are far more specific. 相似文献
13.
Swaney DL McAlister GC Wirtala M Schwartz JC Syka JE Coon JJ 《Analytical chemistry》2007,79(2):477-485
Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%). 相似文献
14.
Huang Y Triscari JM Tseng GC Pasa-Tolic L Lipton MS Smith RD Wysocki VH 《Analytical chemistry》2005,77(18):5800-5813
Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from singly charged peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s) in multiply protonated peptides. Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and may be useful in algorithm development that employs improved models to predict fragment ion intensities. 相似文献
15.
Localization of labile posttranslational modifications by electron capture dissociation: the case of gamma-carboxyglutamic acid. 总被引:5,自引:0,他引:5
N L Kelleher R A Zubarev K Bush B Furie B C Furie F W McLafferty C T Walsh 《Analytical chemistry》1999,71(19):4250-4253
Tandem mass spectrometry (MS/MS) of 28 residue peptides harboring gamma-carboxylated glutamic acid residues, a posttranslational modification of several proenzymes of the blood coagulation cascade, using either collisions or infrared photons results in complete ejection of the gamma-CO2 moieties (-44 Da) before cleavage of peptide-backbone bonds. However, MS/MS using electron capture dissociation (ECD) in a Fourier transform mass spectrometer cleaves backbone bonds without ejecting CO2, allowing direct localization of this labile modification. Sulfated side chains are also retained in ECD backbone fragmentations of a 21-mer peptide, although CAD causes extensive SO3 loss. ECD thus is a unique complement to conventional methods for MS/MS, causing less undesirable loss of side-chain functionalities as well as more desirable backbone cleavages. 相似文献
16.
van Agthoven MA Chiron L Coutouly MA Delsuc MA Rolando C 《Analytical chemistry》2012,84(13):5589-5595
2D FT-ICR MS allows the correlation between precursor and fragment ions by modulating ion cyclotron radii for fragmentation modes with radius-dependent efficiency in the ICR cell without the need for prior ion isolation. This technique has been successfully applied to ion-molecule reactions, Collision-induced dissociation and infrared multiphoton dissociation. In this study, we used electron capture dissociation for 2D FT-ICR MS for the first time, and we recorded two-dimensional mass spectra of peptides and a mixture of glycopeptides that showed fragments that are characteristic of ECD for each of the precursor ions in the sample. We compare the sequence coverage obtained with 2D ECD FT-ICR MS with the sequence coverage obtained with ECD MS/MS and compare the sensitivities of both techniques. We demonstrate how 2D ECD FT-ICR MS can be implemented to identify peptides and glycopeptides for proteomics analysis. 相似文献
17.
Fragmentation of singly protonated peptides via a combination of infrared and collisional activation
The coupling of matrix-assisted laser desorption/ionization (MALDI) to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides an exceptionally capable platform for peptide analysis, but an important limitation of this approach is the difficulty in obtaining informative tandem mass spectra (MS/MS) of singly protonated peptides. This difficulty is especially pronounced with peptide ions containing basic amino acid residues (for example, tryptic peptides). While such ions can be fragmented in some instrument configurations, most FTICR instruments have comparatively little facility for high-energy fragmentation. Here, a novel MS/MS approach implemented with MALDI-FTICR-MS and specifically intended for enhanced fragmentation of singly protonated peptides is described. The method involves infrared irradiation in concert with the simultaneous application of sustained off-resonance irradiation collision-induced dissociation (SORI-CID). This form of MS/MS, described as a combination of infrared and collisional activation (CIRCA), is shown to provide a greater capacity for dissociation of singly charged model peptide ions as compared to infrared multiphoton dissociation (IRMPD) or SORI-CID alone. Overall, the CIRCA approach is demonstrated to be a feasible technique for accessing useful fragmentation pathways of singly charged peptides, including those harboring basic amino acid residues--a crucial feature in the context of proteomics. 相似文献
18.
In hot electron capture dissociation (HECD), multiply protonated polypeptides fragment upon capturing approximately 11-eV electrons. The excess of energy upon the primary c, z* cleavage induces secondary fragmentation in z* fragments. The resultant w ions allow one to distinguish between the isomeric Ile and Leu residues. The analytical utility of HECD is evaluated using tryptic peptides from the bovine milk protein PP3 containing totally 135 amino acid residues. Using a formal procedure for Ile/Leu (Xle) residue assignment, the identities of 20 out of 25 Xle residues (80%) were determined. The identity of an additional two residues could be correctly guessed from the absence of the alternative w ions, and only two residues, for which neither expected nor alternative w ions were observed, remained unassigned. Reinspection of conventional ECD spectra also revealed the presence of Xle w ions, although at lower abundances, with 44% of all Xle residues distinguished. Using a dispenser cathode as an electron source, identification of four out of five Xle residues in a 2.7-kDa peptide was possible with one acquisition 2 s long, with identification of all five residues by averaging of five such acquisitions. Unlike the case of high-energy collision-induced dissociation, no d ions were observed in the HECD of tryptic peptides. 相似文献
19.
The infrared multiple photon dissociation (IRMPD) spectra of O-glycosylated peptides in the gas phase were studied in the IR scanning range of 5.7-9.5 μm. Fragmentation of protonated and sodiated O-glycopeptides was investigated using electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with a free electron laser (FEL). FEL is used in the IRMPD technique as a tunable IR light source. In the IRMPD spectroscopic analysis of the protonated O-glycopeptide, fragment ions of the b/y and B/Y types were observed in the range of 5.7-9.5 μm, corresponding to the cleavage of the backbone in the parent amino acid sequence and glycosyl bonds, whereas the spectra of the sodiated glycopeptide showed major peaks of photoproducts of the B/Y type in the range of 8.4-9.5 μm. The IRMPD spectra of the O-glycopeptides were compared with simulated IR spectra for the structures obtained from the molecular dynamics. 相似文献
20.
Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research. 相似文献