Magnetic Immunoaffinity Enrichment for Selective Capture and MS/MS Analysis of N‑Terminal-TMPP-Labeled Peptides

Proteogenomics is the alliance of proteomics and genomics with the aim of better annotating structural genes based on experimental, protein-based data items established by tandem mass spectrometry. While, on average, more than one-tenth of protein N-termini are incorrectly annotated, there is a crucial need for methodological approaches to systematically establish the translational starts of polypeptides, and their maturations, such as N-terminal methionine processing and peptide signal excision. Refinement of genome annotation through correction of wrongly annotation initiation start site and detection of unannotated genes can be achieved after enrichment and detection of protein N-termini by mass spectrometry. Here we describe a straightforward strategy to specifically label protein N-termini with a positively charged TMPP label to selectively capture these entities with in-house–developed <i>anti</i>-TMPP antibodies coupled to magnetic beads and to analyze them by nanoLC–MS/MS. While most N-terminomics-oriented approaches are based on the depletion of internal peptides to retrieve N-terminal peptides, this enrichment approach is fast and the results are highly specific for improved, ionizable, TMPP-labeled peptides. The whole proteome of the model marine bacterium, <i>Roseobacter denitrificans</i>, was analyzed, leading to the identification of more than twice the number of N-terminal peptides compared with the nonenriched fraction. A total of 269 proteins were characterized in terms of their N-termini. In addition, three unannotated genes were identified based on multiple, redundant N-terminal peptides. Our strategy greatly simplifies the systematic and automatic proteogenomic annotation of genomes as well as degradomics-oriented approaches, focusing the mass spectrometric efforts on the most crucial enriched fractions

A Simplified Label-Free Method for Proteotyping Sets of Six Isolates in a Single Liquid Chromatography-High-Resolution Tandem Mass Spectrometry Analysis

Clinical diagnostics and microbiology require high-throughput identification of microorganisms. Sample multiplexing prior to detection is an attractive means to reduce analysis costs and time-to-result. Recent studies have demonstrated the discriminative power of tandem mass spectrometry-based proteotyping. This technology can rapidly identify the most likely taxonomical position of any microorganism, even uncharacterized organisms. Here, we present a simplified label-free multiplexing method to proteotype isolates by tandem mass spectrometry that can identify six microorganisms in a single 20 min analytical run. The strategy involves the production of peptide fractions with distinct hydrophobicity profiles using spin column fractionation. Assemblages of different fractions can then be analyzed using mass spectrometry. Results are subsequently interpreted based on the hydrophobic characteristics of the peptides detected, which make it possible to link each taxon identified to the initial sample. The methodology was tested on 32 distinct sets of six organisms including several worst-scenario assemblages–with differences in sample quantities or the presence of the same organisms in multiple fractions–and proved to be robust. These results pave the way for the deployment of tandem mass spectrometry-based proteotyping in microbiology laboratories

A Simplified Label-Free Method for Proteotyping Sets of Six Isolates in a Single Liquid Chromatography-High-Resolution Tandem Mass Spectrometry Analysis

Clinical diagnostics and microbiology require high-throughput identification of microorganisms. Sample multiplexing prior to detection is an attractive means to reduce analysis costs and time-to-result. Recent studies have demonstrated the discriminative power of tandem mass spectrometry-based proteotyping. This technology can rapidly identify the most likely taxonomical position of any microorganism, even uncharacterized organisms. Here, we present a simplified label-free multiplexing method to proteotype isolates by tandem mass spectrometry that can identify six microorganisms in a single 20 min analytical run. The strategy involves the production of peptide fractions with distinct hydrophobicity profiles using spin column fractionation. Assemblages of different fractions can then be analyzed using mass spectrometry. Results are subsequently interpreted based on the hydrophobic characteristics of the peptides detected, which make it possible to link each taxon identified to the initial sample. The methodology was tested on 32 distinct sets of six organisms including several worst-scenario assemblages–with differences in sample quantities or the presence of the same organisms in multiple fractions–and proved to be robust. These results pave the way for the deployment of tandem mass spectrometry-based proteotyping in microbiology laboratories

Proteogenomic Definition of Biomarkers for the Large <i>Roseobacter</i> Clade and Application for a Quick Screening of New Environmental Isolates

Whole-cell, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become a routine and reliable method for microbial characterization due to its simplicity, low cost, and high reproducibility. The identification of microbial isolates relies on the spectral resemblance of low-molecular-weight proteins to already-existing isolates within the databases. This is a gold standard for clinicians who have a finite number of well-defined pathogenic strains but represents a problem for environmental microbiologists with an overwhelming number of organisms to be defined. Here we set a milestone for implementing whole-cell MALDI-TOF mass spectrometry to identify isolates from the biosphere. To make this technique accessible for environmental studies, we propose to (i) define biomarkers that will always show up with an intense <i>m</i>/<i>z</i> signal in the MALDI-TOF spectra and (ii) create a database with all the possible <i>m</i>/<i>z</i> values that these biomarkers can generate to screen new isolates. We tested our method with the relevant marine <i>Roseobacter</i> lineage. The use of shotgun nanoLC-MS/MS proteomics on the small proteome fraction of nine <i>Roseobacter</i> strains and the proteogenomic toolbox helped us to identify potential biomarkers in terms of protein abundance and low variability among strains. We show that the DNA binding protein, HU, and the ribosomal proteins, L29 and L30, are the most robust biomarkers within the <i>Roseobacter</i> clade. The molecular weights of these three biomarkers, as for other conserved homologous proteins, vary due to sequence variation above the genus level. Therefore, we calculated the <i>m</i>/<i>z</i> values expected for each one of the known <i>Roseobacter</i> genera and tested our strategy during an extensive screening of natural marine isolates obtained from coastal waters of the Western Mediterranean Sea. The use of this technique versus standard sequencing methods is discussed

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

Proteogenomic Definition of Biomarkers for the Large <i>Roseobacter</i> Clade and Application for a Quick Screening of New Environmental Isolates

Whole-cell, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become a routine and reliable method for microbial characterization due to its simplicity, low cost, and high reproducibility. The identification of microbial isolates relies on the spectral resemblance of low-molecular-weight proteins to already-existing isolates within the databases. This is a gold standard for clinicians who have a finite number of well-defined pathogenic strains but represents a problem for environmental microbiologists with an overwhelming number of organisms to be defined. Here we set a milestone for implementing whole-cell MALDI-TOF mass spectrometry to identify isolates from the biosphere. To make this technique accessible for environmental studies, we propose to (i) define biomarkers that will always show up with an intense <i>m</i>/<i>z</i> signal in the MALDI-TOF spectra and (ii) create a database with all the possible <i>m</i>/<i>z</i> values that these biomarkers can generate to screen new isolates. We tested our method with the relevant marine <i>Roseobacter</i> lineage. The use of shotgun nanoLC-MS/MS proteomics on the small proteome fraction of nine <i>Roseobacter</i> strains and the proteogenomic toolbox helped us to identify potential biomarkers in terms of protein abundance and low variability among strains. We show that the DNA binding protein, HU, and the ribosomal proteins, L29 and L30, are the most robust biomarkers within the <i>Roseobacter</i> clade. The molecular weights of these three biomarkers, as for other conserved homologous proteins, vary due to sequence variation above the genus level. Therefore, we calculated the <i>m</i>/<i>z</i> values expected for each one of the known <i>Roseobacter</i> genera and tested our strategy during an extensive screening of natural marine isolates obtained from coastal waters of the Western Mediterranean Sea. The use of this technique versus standard sequencing methods is discussed

Proteogenomic Definition of Biomarkers for the Large <i>Roseobacter</i> Clade and Application for a Quick Screening of New Environmental Isolates

Whole-cell, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become a routine and reliable method for microbial characterization due to its simplicity, low cost, and high reproducibility. The identification of microbial isolates relies on the spectral resemblance of low-molecular-weight proteins to already-existing isolates within the databases. This is a gold standard for clinicians who have a finite number of well-defined pathogenic strains but represents a problem for environmental microbiologists with an overwhelming number of organisms to be defined. Here we set a milestone for implementing whole-cell MALDI-TOF mass spectrometry to identify isolates from the biosphere. To make this technique accessible for environmental studies, we propose to (i) define biomarkers that will always show up with an intense <i>m</i>/<i>z</i> signal in the MALDI-TOF spectra and (ii) create a database with all the possible <i>m</i>/<i>z</i> values that these biomarkers can generate to screen new isolates. We tested our method with the relevant marine <i>Roseobacter</i> lineage. The use of shotgun nanoLC-MS/MS proteomics on the small proteome fraction of nine <i>Roseobacter</i> strains and the proteogenomic toolbox helped us to identify potential biomarkers in terms of protein abundance and low variability among strains. We show that the DNA binding protein, HU, and the ribosomal proteins, L29 and L30, are the most robust biomarkers within the <i>Roseobacter</i> clade. The molecular weights of these three biomarkers, as for other conserved homologous proteins, vary due to sequence variation above the genus level. Therefore, we calculated the <i>m</i>/<i>z</i> values expected for each one of the known <i>Roseobacter</i> genera and tested our strategy during an extensive screening of natural marine isolates obtained from coastal waters of the Western Mediterranean Sea. The use of this technique versus standard sequencing methods is discussed

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

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

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