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

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

Saturation experiments with ¹²⁵I-HEAT on receptor variants.

<p>Saturation experiments with ¹²⁵I-HEAT on receptor variants.</p

Pharmacological profile of ρ-Da1a binding to various human AR subtypes expressed in eukaryotic cells.

<p>Binding inhibition curves for ³H-prazosin (2 nM), ³H-rauwolscine (2 nM) and ³H-CGP-12177 (6 nM) on hα_1A- (1 µg, ○), hα_1B- (3 µg, •), hα_1D- (29 µg, □), hα_2A- (140 µg, ◊), hα_2B- (100 µg, Δ), hα_2C- (3 µg, x), β₁- (3 µg,▾) and β₂-AR (1.5 µg, ▪) with recombinant ρ-Da1a. n = 4.</p

Receptor affinities for ρ-Da1a (dash lines) and HEAT (solid lines) on mutated α_1A-ARs.

<p>Binding inhibition curves for ¹²⁵I-HEAT binding to WT (200 pM, 0.2 µg, ○), D106^3.32A (200 pM, 1 µg, □) and F86^2.64A (1.3 nM, 0.8 µg, •) receptor variants. n = 3–4.</p

Influence of various ligands on ³H-prazosin and ¹²⁵I-HEAT dissociation.

<p>Panel A: Dissociation of ³H-prazosin (2 nM) binding to α_1A-AR (1 µg) in the presence of prazosin (10 µM, black), prazosin plus ρ-Da1a (2.5 µM, blue), prazosin plus adrenaline (2 mM, red) and prazosin plus EPA (150 µM, green). Panel B : dissociation of ¹²⁵I-HEAT (0.4 nM) binding to α_1A-AR (0.2 µg) in the presence of HEAT (5 µM, black), HEAT plus ρ-Da1a (2.5 µM, blue), HEAT plus prazosin (10 µM, red) and HEAT plus EPA (150 µM, green). n = 2.</p

Inhibition of the binding of a series of concentrations of ³H-prazosin and ¹²⁵I-HEAT to α_1A-AR by ρ-Da1a.

<p>Panel A ³H-prazosin binding (from 0.2 to 16 nM) inhibited by ρ-Da1a. Panel B ¹²⁵I-HEAT binding (from 0.1 to 1.25 nM) inhibited by ρ-Da1a. Panel C and D: Fitting, by the Cheng and Prusoff equation IC₅₀ = Ki+Ki(L/Kd), of IC₅₀ values as a function of the radiotracer concentrations.</p

Effect of human α_1A-AR mutations on receptor expression and affinity for HEAT and ρ-Da1a.

*<p>for p&lt;0.05. Position refers to the Ballesteros-Weinstein numbering scheme for residues within TM domains of G protein-coupled receptors. n = 3–6.</p

Homology modelling of the ρ-Da1a binding site in the α_1A-AR and the MT7 toxin.

<p>Views from the side of the TM bundle (Panel A), and from the top of the extracellular space (Panel B). F187^5.41, F193^5.47, F281^6.44, M292^6.55, F308^7.35 in green. D106^3.32 and the double S188^5.42/S192^5.46 in orange. F86^2.64, F288^6.51 and F312^7.39 in red. Panel C :3D structure of the three-finger fold MT7 toxin (2vlw) with the four conserved disulfide bridges in red.</p

Inhibition of ³H-prazosin (2 nM, 1 µg, open symbols) by HEAT (□), and ρ-Da1a (circle), and inhibition of ¹²⁵I-HEAT (0.2 nM, 0.2 µg, full symbols) binding by prazosin (♦) and ρ-Da1a (circle) to α_1A-AR.

<p>n = 3.</p