Liquid Chromatography Electron Capture Dissociation Tandem Mass Spectrometry (LC-ECD-MS/MS) versus Liquid Chromatography Collision-induced Dissociation Tandem Mass Spectrometry (LC-CID-MS/MS) for the Identification of Proteins

Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins.Experiments were performed on a hybrid linear ion trap–Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin,lysozyme, cytochrome c, alcohol dehydrogenase, and β-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECDMS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags,providing greater confidence in protein assignment

Efficiency of Collisionally-activated dissociation and 193-nm photodissociation of peptide ions in fourier transform mass spectrometry

AbstractFor tandem mass spectrometry, the Fourier transform instrument exhibits advantages for the use of collisionally-activated dissociation (CAD). The CAD energy deposited in larger ions can be greatly increased by extending the collision time to as much as 120 s, and the efficiency of trapping and measuring CAD product ions in many times greater than the found for triple-quadrupole or magnetic sector instruments, although the increased pressure from the collision gas is an offsetting disadvantage. A novel system that uses the same laser for photodesorption of ions and their subsequent photodissociation can produce complete dissociation of larger oligopeptide ions and unusually abundant fragment ions. In comparison to CAD, much more internal energy can be deposited in the primary ions using 193-nm photons, sufficient to dissociate peptide ions of m/z > 2000. Mass spectra closely resembling ion photodissociation spectra can also be obtained by neutral photodissociation (193-nm laser irradiation of the sample) followed by ion photodesorption

Effects of Charge State and Cationizing Agent on the Electron Capture Dissociation of a Peptide

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. Although mass spectrometry (MS) and tandem mass spectrometry (MS/MS) have been used to characterize peptides for more than three decades, 1,2 the developments of electrospray ionization (ESI) 3 and matrix-assisted laser desorption/ionization 4 have dramatically expanded the size and type of molecules amenable to characterization by MS/MS. For example, ESI has been used to form intact gas-phase ions from virus particles (4.0 × 10 7 Da) 5 and DNA molecules as large as 1.2 × 10 8 Da. 6 ESI-MS and ESI-MS/MS experiments can be performed using as little as 10 -18 mol of sample. 7 For these measurements, Fourier transform (FT) MS has the advantages of ultrahigh resolution, multichannel detection, and MS n capabilities. 8,9 Dissociation methods in FTMS, including collisionally activated dissociation (CAD), 10 surface-induced dissociation, 11,12 infrared multiphoton dissociation, 13 and blackbody infrared radiative dissociation, 14,15 have been used to obtain sequence information and locations of posttranslational modifications (PTMs) in biomolecules. With these activation methods, the most labile bonds within an ion are typically cleaved. This often produces incomplete sequence coverage, the loss of PTMs, and a lack of backbone cleavages within regions enclosed by disulfide bridges. The recently developed method of electron capture dissociation (ECD), [16][17][18][19][20][21][22][23][24][25