Dissociation mechanisms and implication for the presence of multiple conformations for peptide ions with arginine at the C‐terminus: time‐resolved photodissociation study

Time‐resolved photodissociation (PD) patterns of singly protonated peptides with arginine at the C‐terminus (C‐arg peptide ions) have been used to classify the dissociation channels into two categories, i.e. high‐energy channels generating v, w and x and low‐energy ones generating b, y and z. x + 1 formed by C_α? CO cleavage seems to be the intermediate ion in high‐energy channels just as a + 1 is for N‐arg peptide ions. Difference in time‐resolved pattern indicates that the two sets of channels, high‐ and low‐energy ones, are not in direct competition. Noncompetitive dissociation is also indicated by the observation of anomalous effect of matrix used in matrix‐assisted laser desorption ionization, a cooler matrix generating more high‐energy product ions both in spontaneous dissociation and in PD. Results from detailed investigation suggest that the two sets of channels start from two (or more) different conformations. Copyright © 2010 John Wiley & Sons, Ltd.     

The use of proteotypic peptide libraries for protein identification

This paper describes an algorithm to apply proteotypic peptide sequence libraries to protein identifications performed using tandem mass spectrometry (MS/MS). Proteotypic peptides are those peptides in a protein sequence that are most likely to be confidently observed by current MS-based proteomics methods. Libraries of proteotypic peptide sequences were compiled from the Global Proteome Machine Database for Homo sapiens and Saccharomyces cerevisiae model species proteomes. These libraries were used to scan through collections of tandem mass spectra to discover which proteins were represented by the data sets, followed by detailed analysis of the spectra with the full protein sequences corresponding to the discovered proteotypic peptides. This algorithm (Proteotypic Peptide Profiling, or P3) resulted in sequence-to-spectrum matches comparable to those obtained by conventional protein identification algorithms using only full protein sequences, with a 20-fold reduction in the time required to perform the identification calculations. The proteotypic peptide libraries, the open source code for the implementation of the search algorithm and a website for using the software have been made freely available. Approximately 4% of the residues in the H. sapiens proteome were required in the proteotypic peptide library to successfully identify proteins.     

Gas‐phase decomposition mechanisms of C‐NO2, N‐NO2 energetic materials: Reevaluations

The thermal decomposition mechanisms of gaseous nitromethane, methyl nitrite, dimethylnitramine and 1,3,3,‐trinitroazetidine (TNAZ) have been reanalyzed using sensitivity and principal component analyses. For each system an adequate, much simplified mechanism was developed, and the fragmentation/reaction sequence leading to the final products was made transparent. The critical roles of free radicals (CH₃, H, OH, HCO, HNO, CH₃O, etc.) at different stages of these pyrolyses were then identified. The predicted product distributions for these compounds were calculated to facilitate the assessment of their explosive performance parameters. Comparison of the products generated by nitromethane with those produced by methyl nitrite showed that the contribution of a nitro‐nitrite isomerization reaction is negligible in the nitromethane pyrolysis. © 1999 John Wiley & Sons, Inc. Int J Chem Kinet 31: 655–673, 1999     

Improvement of gel‐separated protein identification by DMF‐assisted digestion and peptide recovery after electroblotting

In‐gel digestion of gel‐separated proteins is a major route to assist in proteomics‐based biological discovery, which, however, is often embarrassed by its inherent limitations such as the low digestion efficiency and the low recovery of proteolytic peptides. For overcoming these limitations, many efforts have been directed at developing alternative methods to avoid the in‐digestion. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel‐separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (≥99.8%). Before tryptic digestion, NH₄HCO₃ buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane‐bound substances, the proteins were virtually in‐solution digested in DMF‐containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane‐enriched sample showed that the method was superior to the reported on‐membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.     

Characterizing protein glycosylation sites through higher‐energy C‐trap dissociation

Assigning glycosylation sites of glycoproteins and their microheterogeneity is still a very challenging analytical task despite the rapid advancements in mass spectrometry. It is shown here that glycopeptide ions can be fragmented efficiently using the higher‐energy C‐trap dissociation (HCD) feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap). An attractive aspect of this dissociation option is the generation of distinct Y1 ions (peptide+GlcNAc), thus allowing unequivocal assignment of N‐glycosylation sites of glycoproteins. The combination of the very informative collision‐induced dissociation spectra acquired in the linear ion trap with the distinct features of HCD offers very useful information aiding in the characterization of the glycosylation sites of glycoproteins. The HCD activation energy needed to obtain optimum Y1 ions was studied in terms of glycan structure and charge state, and size and structure of the peptide backbone. The latter appeared to be primarily dictating the needed HCD energy. The distinct Y1 ion formation in HCD facilitated an easy assignment of such an ion and its subsequent isolation and dissociation through multiple‐stage tandem mass spectrometry. The resulting MS³ spectrum of the Y1 ion facilitates database searching and de novo sequencing thus prompting the subsequent identification of the peptide backbone and associated glycosylation sites. Moreover, fragment ions formed by HCD are detected in the Orbitrap, thus overcoming the 1/3 cut‐off limitation that is commonly associated with ion trap mass spectrometers. As a result, in addition to the Y1 ion, the common glycan oxonium ions are also detected. The high mass accuracy offered by the LTQ Orbitrap mass spectrometer is also an attractive feature that allows a confident assignment of protein glycosylation sites and the microheterogeneity of such sites. Copyright © 2010 John Wiley & Sons, Ltd.