Artifacts and unassigned masses encountered in peptide mass mapping

Self Cite

The fragmentation of 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid [leukotriene C 4 or LTC 4 (5, 6)] and its isomeric counterpart LTC 4 (14, 15) were studied by low and high-energy collisional induced dissociation (CID) and 157 nm photofragmentation. For singly charged protonated LTC 4 precursors, photodissociation significantly enhances the signal intensities of informative fragment ions that are very important to distinguish the two LTC 4 isomers and generates a few additional fragment ions that are not usually observed in CID experiments. The ion trap enables MS n experiments on the fragment ions generated by photodissociation. Photofragmentation is found to be suitable for the structural identification and isomeric differentiation of cysteinyl leukotrienes and is more informative than low or high-energy CID. We describe for the first time the structural characterization of the LTC 4 (14, 15) isomer by mass spectrometry using CID and 157 nm light activation methods. (J

Section: Protonated Ltc 4 (5 6) Isomermentioning

confidence: 99%

mentioning

confidence: 99%

Structural analysis of leukotriene C₄isomers using collisional activation and 157 nm photodissociation

Devakumar

O'Dell

Walker

et al. 2008

Self Cite

“…It has been observed that in contrast to other molecules, for peptides the first decimal place of the mass is near the half of the first digit of mass values between 500 and 999 and near the half of the first two digits of mass values between 1000 and 1999 [25,26]. In the mass range 2000 to 3000 again the first decimal place is near the half of the first digit of the peptide mass.…”

Section: Characteristics Of Peptide Masses: the Halfdecimal Place Rulementioning

confidence: 99%

Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis

Schmidt

Schmid

Jungblut

et al. 2003

Peptide mass fingerprinting (PMF) is a powerful tool for identification of proteins separated by two-dimensional electrophoresis (2-DE). With the increase in sensitivity of peptide mass determination it becomes obvious that even spots looking well separated on a 2-DE gel may consist of several proteins. As a result the number of mass peaks in PMFs increased dramatically leaving many unassigned after a first database search. A number of these are caused by experiment-specific contaminants or by neighbor spots, as well as by additional proteins or post-translational modifications. To understand the complete protein composition of a spot we suggest an iterative procedure based on large numbers of PMFs, exemplified by PMFs of 480 Helicobacter pylori protein spots. Three key iterations were applied: (1) Elimination of contaminant mass peaks determined by MS-Screener (a software developed for this purpose) followed by reanalysis; (2) neighbor spot mass peak determination by cluster analysis, elimination from the peak list and repeated search; (3) re-evaluation of contaminant peaks. The quality of the identification was improved and spots previously unidentified were assigned to proteins. Eight additional spots were identified with this procedure, increasing the total number of identified spots to 455. [5][6][7], represent major milestones on the way to an understanding of the proteome. A further step was the application of mass spectrometry for peptide mass fingerprinting (PMF) instead of SDS gel electrophoresis [8], which resulted in a quick and reliable identification of 2-DE separated protein spots [9,10]. Instead of measuring the mass of the uncleaved proteins the high mass accuracy of MALDI-and ESI-MS in the Mr range between 500 and 3000 was utilized.PMF as a probability method is especially powerful if the data set of potential protein candidates is small, as in the case of microorganisms with completely sequenced genomes. For unequivocal identification of post-translational modifications, complementation by protein sequencing methods is necessary. PMF has been used successfully for the identification of several hundred proteins of Haemophilus influenzae [11], Mycobacterium tuberculosis [12], Helicobacter pylori [13], and Mycoplasma pneumoniae [14]. These investigations clearly demonstrated that proteins often occur as different protein species at different positions within the 2-DE gel. However, one spot may also represent several

“…However, at lower analyte concentrations only a signal at m/z 1066.1 could be observed (Figure 2c-f). The fractional part of peptide masses can be predicted [19] and usually ranges from 0.4 to 0.7 Da for peptides between 1000 and 1100 Da [11]. This is a result of the contribution of near-integral masses of the elements typically found in peptides, such as C, H, N, O, and S [10,19].…”

Section: Maldi Tof Mass Spectrometry Using Chcamentioning

confidence: 99%

“…Nonetheless, these signals may complicate data analysis by increased spectral complexity but their value as internal mass calibrants in MALDI TOF mass spectrometry has been recognized [10]. Moreover, knowledge about matrix cluster masses may also be important when analyzing protein digests in order to exclude these signals from database searches for enhanced confidence scores [11].…”

mentioning

confidence: 99%

MALDI post-source decay and LIFT-TOF/TOF investigation of α-cyano-4-hydroxycinnamic acid cluster interferences

Neubert

Halket

Ocaña

et al. 2004

Large signals from ␣-cyano-4-hydroxycinnamic acid (CHCA) matrix complexes with sodium and potassium ions were found to interfere with sensitive matrix-assisted laser desorption/ ionization (MALDI) analysis of a hydrochloric acid digest of gelatine preparations. The nature of some selected matrix clusters was investigated by conventional post-source decay and LIFT-TOF/TOF experiments. The matrix clusters fragmented readily by neutral evaporation to give smaller sized matrix cluster species without matrix disintegration. Their characterization distinguished them from peptide signals, in particular from those that had the same nominal mass and differed only in the fractional part of the mass as encountered for gelatine-derived peptides. Knowledge of the molecular composition of these cluster species allowed using them for internal calibration of the MALDI mass spectra. The hydrolytic peptides could be analyzed with increased sensitivity when using 2,5-dihydroxy benzoic acid (DHB) as the MALDI matrix. (J Am Soc Mass Spectrom 2004, 15, 336 -343)