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Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes

ABSTRACT: Datasets part of manuscript submitted at analytica chimica acta Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.0 mm internal diameter, I.D.) operated at 15-uL/min flow rates and their coupling to nMS for the characterization of proteins, protein complexes and higher-order structures (HOS). The reduced flow rate resulted in a significant increase in the protein-ionization efficiency, facilitating the detection of low-abundant impurities and HOS up to 230 kDa (i.e., the upper limit of the Orbitrap-MS instrument used). More-efficient solvent evaporation and lower desolvation energies allowed for softer ionization conditions (e.g., lower gas temperatures), ensuring little or no structural alterations of proteins and their HOS during transfer into the gas phase. Furthermore, ionization suppression by eluent salts was decreased, permitting the use of volatile-salt concentrations up to 400 mM. Band broadening and loss of resolution resulting from the introduction of injection volumes exceeding 3% of the column volume could be circumvented by incorporating an online trap-column containing a mixed-bed ion-exchange (IEX) material. The online IEX-based solid-phase extraction (SPE) or trap-and-elute set-up provided on-column focusing (sample preconcentration). This allowed the injection of large sample volumes on the 1-mm I.D. SEC column without compromising the separation. The enhanced sensitivity attained by the micro-flow SEC-MS, along with the on-column focusing achieved by the IEX precolumn, provided picogram detection limits for proteins.

INSTRUMENT(S): QExactive biopharma

ORGANISM(S): Reference Proteins

SUBMITTER: Andrea Gargano

PROVIDER: MSV000091582 | MassIVE | Wed Mar 29 01:43:00 BST 2023

REPOSITORIES: MassIVE

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Project description:In this study we analyzed and compared total proteome and secretome of the wild-type and JN1 mutant strains of Bordetella pertussis. The single-nucleotide transversion in the 5’-UTR of the rplN gene of JN1 mutant led to the increased transcription of the whole operon encoding ribosomal proteins and of the adjoining rpoA gene. These events led to the downregulation and decreased secretion of virulence factors on the background ofgenerally deregulated expression of B. pertussis genome. To get deeper inside in the molecular mechanisms of the observed genome deregulation we then performed the immunoprecipitation of RpoA and compared its binding partners in wild-type and JN1 mutant strains. Nano Reversed phase column (EASY-Spray column, 50 cm x 75 µm ID, PepMap C18, 2µm particles, 100Å pore size) was used for LC/MS analysis. Mobile phase buffer A was composed of water and 0.1% formic acid. Mobile phase B was composed of acetonitrile and 0.1% formic acid. Samples were loaded onto the trap column (Acclaim PepMap300, C18, 5µm, 300Å wide Pore, 300 µm x 5 mm) at a flow rate of 15 μl/min. Loading buffer was composed of water, 2% acetonitrile and 0.1% trifluoroacetic acid. Peptides were eluted with gradient of B from 4% to 35% over 60 min at a flow rate of 300 nl/min. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT- qIT, Thermo). Survey scans of peptide precursors from 350 to 1400 m/z were performed at 120K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 1.5 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS 2 ion count target was set to 104 and the max injection time was 35 ms. Only those precursors with the charge state 2–6 were sampled for MS 2. The dynamic exclusion duration was set to 45 s with a 10ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 2s cycles (76). The data were analyzed and quantified with label-free quantification (LFQ) algorithms in MaxQuant v1.6.3.3 (77) and the Andromeda search engine(78). The false discovery rate (FDR) parameter was set to 1 % for both proteins and peptides. The enzyme specificity of trypsin was set as C-terminal to Arg and Lys. Carbamidomethylation was set as the fixed modification, while N-terminal protein acetylation and methionine oxidation were variable modifications. Maximal number of missed cleavages was set to 2. All hits identified in searches as contaminants were filtered out. The data were searched against Bordetella pertussis reference proteome database (strain Tohama I / ATCC BAA-589 / NCTC 13251).

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Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes

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