MS/MS spectrum of the semi-tryptic peptide [288–299] belonging to ZP_02147451.

<p>The MS/MS spectrum was acquired with a FT/FT procedure with an LTQ-Orbitrap XL hybrid mass spectrometer. The peptide sequence is shown on the top with the collision-induced fragmentation pattern. The <i>b</i> and <i>y</i> ions are shown in blue and red, respectively. The <i>y₁₁</i> di-charged ion is labeled in green.</p

Occurrence of basic residues in proteins from <i>Phaeobacter strain DSM 17395.</i>

<p>The graph reports the ratio Lysine (K) and Arginine (R) residues per protein length of all the proteins with length above 100 residues encoded by <i>Phaeobacter strain DSM 17395</i>. Proteins are symbolized by a blue triangle. RTX-like proteins and the other exoproteins detected by tandem mass spectrometry are represented by red and yellow squares, respectively. The ZP_02143988.1 (a), ZP_02147451.1 (b), ZP_02144693.1 (c), ZP_02146960.1 (d), ZP_02144235.1 (e), and ZP_02146514.1 (f) RTX-toxins are indicated. Only the ZP_02143988.1 RTX-like protein has been detected by mass spectrometry.</p

ZP_02147451 sequence coverage with non-tryptic, semi-tryptic, and tryptic peptides.

<p>The ZP_02147451 sequence is represented with its peptidase motif (residues 240 to 279) pointed out with purple stars. Peptides identified with the “no-enzyme” Mascot search are symbolized with a line underlining the sequence. Tryptic, semi-tryptic, and non-tryptic peptides are indicated in yellow, blue and red, respectively.</p

SDS-PAGE of the exoproteome of <i>Phaeobacter strain DSM 17395</i>.

<p>Exoproteins were resolved by a long migration on a 10% SDS-PAGE and stained with SimplyBlue SafeStain (Invitrogen). Lane <b>M</b>: SeeBlue Plus2 molecular weight range marker (Invitrogen). Lane <b>E1</b>: <i>Phaeobacter strain DSM 17395</i> exoproteome grown in Marine Broth (20 µg). Lane <b>E2</b>: <i>Phaeobacter strain DSM 17395</i> exoproteome grown in Marine Broth (8 µg). The 55 kDa major component is indicated with an arrow.</p

List of the first ten proteins identified from the 55<i>P. gallaeciensis</i> DSM¹.

1<p>Detected with at least three different peptides.</p><p>*Periplasmic component.</p

List of proteins identified in the 55/FT proteomic procedure¹.

1<p>Detected with at least three different peptides. The values are from a representative experiment.</p><p>*Periplasmic component.</p>$<p>EUF stands for Enzyme Unspecificity Factor, defined as the ratio number of unique peptides (no-enzyme MASCOT search) per number of unique peptides (trypsin MASCOT search).</p

Proteogenomic Biomarkers for Identification of <i>Francisella</i> Species and Subspecies by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

<i>Francisella tularensis</i> is the causative agent of tularemia. Because some <i>Francisella</i> strains are very virulent, this species is considered by the Centers for Disease Control and Prevention to be a potential category A bioweapon. A mass spectrometry method to quickly and robustly distinguish between virulent and nonvirulent <i>Francisella</i> strains is desirable. A combination of shotgun proteomics and whole-cell matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry on the <i>Francisella tularensis</i> subsp. <i>holarctica</i> LVS defined three protein biomarkers that allow such discrimination: the histone-like protein HU form B, the 10 kDa chaperonin Cpn10, and the 50S ribosomal protein L24. We established that their combined detection by whole-cell MALDI-TOF spectrum could enable (i) the identification of <i>Francisella</i> species, and (ii) the prediction of their virulence level, i.e., gain of a taxonomical level with the identification of <i>Francisella tularensis</i> subspecies. The detection of these biomarkers by MALDI-TOF mass spectrometry is straightforward because of their abundance and the absence of other abundant protein species closely related in terms of <i>m</i>/<i>z</i>. The predicted molecular weights for the three biomarkers and their presence as intense peaks were confirmed with MALDI-TOF/MS spectra acquired on <i>Francisella philomiragia</i> ATCC 25015 and on <i>Francisella tularensis</i> subsp. <i>tularensis</i> CCUG 2112, the most virulent <i>Francisella</i> subspecies