Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation Patterns

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

A Data-Mining Scheme for Identifying Peptide Structural Motifs Responsible for Different MS/MS Fragmentation Intensity Patterns

Although tandem mass spectrometry (MS/MS) has become an integral part of proteomics, intensity patterns in MS/MS spectra are rarely weighted heavily in most widely used algorithms because they are not yet fully understood. Here a knowledge mining approach is demonstrated to discover fragmentation intensity patterns and elucidate the chemical factors behind such patterns. Fragmentation intensity information from 28 330 ion trap peptide MS/MS spectra of different charge states and sequences went through unsupervised clustering using a penalized K-means algorithm. Without any prior chemistry assumptions, four clusters with distinctive fragmentation patterns were obtained. A decision tree was generated to investigate peptide sequence motif and charge state status that caused these fragmentation patterns. This data-mining scheme is generally applicable for any large data sets. It bypasses the common prior knowledge constraints and reports on the overall peptide fragmentation behavior. It improves the understanding of gas-phase peptide dissociation and provides a foundation for new or improved protein identification algorithms

Enrichment of Integral Membrane Proteins for Proteomic Analysis Using Liquid Chromatography−Tandem Mass Spectrometry

An increasing number of proteomic strategies rely on liquid chromatography−tandem mass spectrometry (LC−MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents and chaotropes that often interfere with chromatographic separation and/or electrospray ionization. To address this problem, a sample preparation method combining carbonate extraction, surfactant-free organic solvent-assisted solubilization, and proteolysis was developed and demonstrated to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic on the basis of their calculated hydropathy values (GRAVY index), corresponding to coverage of 15% of the predicted hydrophobic proteome. Using the PSORT algorithm, 53 of the proteins identified were classified as integral outer membrane proteins and 215 were classified as integral cytoplasmic membrane proteins. All identified integral cytoplasmic membrane proteins had from 1 to 16 mapped transmembrane domains (TMDs), and 65% of those containing four or more mapped TMDs were identified by at least one hydrophobic membrane spanning peptide. The extensive coverage of the membrane subproteome (24%) by identification of highly hydrophobic proteins containing multiple TMDs validates the efficacy of the described sample preparation technique to isolate and solubilize hydrophobic integral membrane proteins from complex protein mixtures.Keywords: membrane proteins • sample preparation • hydrophobic proteins • proteomics • mass spectrometr

Dissociation Behavior of Doubly-Charged Tryptic Peptides: Correlation of Gas-Phase Cleavage Abundance with Ramachandran Plots

Analysis of fragmentation patterns from 5654 unique doubly charged tryptic peptides is obtained. Great variability of average relative abundance of bond cleavage is found between different amino acid combinations. There exist similarities as well as differences between b and y ions. Strong enhancement or suppression of cleavage gives insight into possible chemical interactions at reactive conformations formed by preferred phi−psi angles

Characteristics of <i>T. maritima</i> OmpB orthologs and putative syntenic analogs in Thermotogales species.

<p>Characteristics are defined in the text. <b>Legend:</b> +, protein is predicted to possesses the attribute; –, protein is predicted to not possess the attribute.</p><p>ORFs are from TM, T. maritima; TRQ2, Thermotoga species strain RQ2; Tpet, Thermotoga petrophila; Tnap, Thermotoga naphthophila; CTN, Thermotoga neapolitana; Tmel, Thermotoga melanesiensis; Fnod, Fervidobacterium nodosum; Tlet, Thermotoga lettingae; THA, Thermosipho africanus; Kole, Kosmotoga olearia; Pmob, Petrotoga mobilis; and Theba, Thermotogales bacterium mesG1.Ag.4.2 (Mesotoga prima). ORFs above the line are homologs of TM0476, those below the line are possible analogs of TM0476.</p>‡<p>Values of the percent β strand content were calculated utilizing the multisequence alignment programs STRAP and SCRATCH, respectively.</p

The C-terminal amino acids of TM0476 are like those of several confirmed porins.

<p>Position 1 indicates the C-terminal amino acid. Hydrophobic residues are boldface. All porins shown possess the essential terminal phenylalanine and hydrophobic residues at positions 3, 5, 7 and 9.</p

The C-terminal amino acids of the OmpB orthologs and putative analogs show porin characters.

<p>Position 1 indicates the C-terminal amino acid. Hydrophobic residues are boldface. All porins shown possess the essential terminal phenylalanine and most have hydrophobic residues at positions 3, 5, 7 and 9. ORFs above the line are homologs of TM0476, those below the line are possible analogs of TM0476.</p

Syntenic regions containing <i>T. maritima</i> OmpA1 and OmpB homologs and putative analogs mapped onto an rRNA gene reference tree.

<p>The coloring of the individual genes indicates whether the gene is a homolog, paralog, or putative analog. <i>segG</i> homologs are yellow, <i>tyrS</i> homologs are light blue, <i>ompA1</i> homologues are red, and <i>ompB</i> homologues are dark blue. <i>ompA</i> paralogs are white (see Fig. 2). <i>ompB</i> putative analogs are grey. The tree is a concatenated 23S-16S rRNA gene cladogram. Branch lengths do not reflect the extent of divergence.</p

Characteristics of porin candidates from <i>T. maritima</i> as compared to those of known porins.

<p>Characteristics are defined in the text. <b>Legend:</b> +, protein is predicted to possesses the attribute; –, protein is predicted to not possess the attribute.</p>*<p>Proteins are <i>Escherichia coli</i> OmpC, OmpF, PhoE; <i>Salmonella enterica</i> ScrY; and <i>Yersinia enterocolitica</i> ScrY. ORFs are from TM, <i>T. maritima</i>. ‡ Percent β strand content calculated utilizing the multisequence alignment programs STRAP and SCRATCH, respectively.</p

Maximum likelihood phylogenetic tree of OmpA protein sequences from several Thermotogales species.

<p>Red labels indicate likely <i>T. maritima</i> OmpA1 (TM0477) orthologs, green labels indicate likely OmpA2 (TM1729) orthologs, and homologs whose type of homology cannot be ascertained are labeled in black. The tree was calculated as unrooted phylogeny, but is depicted as rooted between the likely OmpA1 and OmpA2 clusters. Branches with approximate Likelihood Ratio Test (aLRT) support values ≤0.75 were collapsed. aLRT, posterior probability, and bootstrap support values are given above or below the branch to which they pertain. Organism abbreviations and gene identification numbers for loci are <i>Petrotoga mobilis</i> (Pmob_0057, 160901548 and Pmob_1624, 160903060); <i>T. lettingae</i> (Tlet_1719, 157364570 and Tlet_0301, 157363168); <i>T. petrophila</i> (Tpet_1024, 148270158 and Tpet_0443, 148269583); <i>T. maritima</i> (TM0477, 15643243 and TM1729, 15644475); <i>T. napthophila</i> (Tnap_1078, 281412500 and Tnap_0259, 281411698) <i>T. neapolitana</i> (CTN_0195, 222099169 and CTN_0927, 222099901); <i>Thermotoga</i>. sp. strain RQ2 (TRQ2_0458, 170288259 and TRQ2_1096, 170288887); <i>Fervidobacterium nodosum</i> (Fnod_1724, 154250391 and Fnod_0047, 154248750); <i>Kosmotoga olearia</i> (Kole_0210, 239616617 and Kole_1500, 239617873); <i>Thermosipho africanus</i> (THA_407, 217076525 and THA_93, 217076226); <i>Thermosipho melanesiensis</i> (Tmel_0176, 150020084 and Tmel_1771, 150021641); Thermotogales bacterium mesG.Ag.4 (<i>Mesotoga prima</i>) (ThebaDRAFT_0522, 307297745; now Theba_0318).</p