A Proteomic Signature of Dormancy in the Actinobacterium: Micrococcus luteus

Dormancy is a protective state in which diverse pathogenic and non-pathogenic bacteria curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states including viable-but-non-culturable (VBNC) and persistence states that contribute to the antibiotic tolerance. Reactivation from latent infection are observed in many serious pathogens including Mycobacterium turberculosis, Staphylococcus, Streptococcus, and Borrelia bacteria. Despite the obvious threat presented by dormant bacteria, the protein mechanisms regulating these dormancy states are not well understood. We have studied VBNC dormancy in Micrococcus luteus NCTC 2665 by tandem mass spectrometry-based quantitative proteomics to uncover some of these mechanisms. M. luteus is a nonpathogenic actinobacterium exhibiting a uniquely well-defined and reproducible VBNC state induced by nutrient deprivation. Dormant M. luteus demonstrated a global loss of protein diversity accompanied by increased levels of eighteen proteins that are conserved across actinobacteria including M. tuberculosis. Four of these proteins have been previously associated with latent tuberculosis, but the other 14 proteins are novel protein targets for dormancy studies. We have developed rapid methods to quantitate dormancy-related proteins across growth phases by targeted proteomics. The proteins upregulated during dormancy implicate important roles for anaplerotic metabolism, redox and amino acid metabolism, ribosomal regulatory processes, and nucleoid associated proteins in dormancy. Our data show that M. luteus is a viable model system for dissecting the protein mechanisms underlying dormancy and we identified new protein targets for future studies on therapeutics active against dormant bacterial infections, which is a severe limitation of current antibiotics.Biology and Biochemistry, Department o

ATG8-Binding UIM Proteins Define a New Class of Autophagy Adaptors and Receptors

© 2019 Elsevier Inc. Across organisms, the key autophagy protein ATG8/LC3 binds a group of proteins on a site distinct from its classical interacting region, raising the possibility of dual binding interactions and identifying an array of previously unknown selective autophagic adaptors and receptors, including some involved in human disease

Enhancing MALDI Time-Of-Flight Mass Spectrometer Performance through Spectrum Averaging

<div><p>Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometers are simple and robust mass spectrometers used for analysis of biologically relevant molecules in diverse fields including pathogen identification, imaging mass spectrometry, and natural products chemistry. Despite high nominal resolution and accuracy, we have observed significant variability where 30–50% of individual replicate measurements have errors in excess of 5 parts-per-million, even when using 5-point internal calibration. Increasing the number of laser shots for each spectrum did not resolve this observed variability. What is responsible for our observed variation? Using a modern MALDI-TOF/TOF instrument, we evaluated contributions to variability. Our data suggest a major component of variability is binning of the raw flight time data by the electronics and clock speed of the analog-to-digital (AD) detection system, which requires interpolation by automated peak fitting algorithms and impacts both calibration and the observed mass spectrum. Importantly, the variation observed is predominantly normal in distribution, which implies multiple components contribute to the observed variation and suggests a method to mitigate this variability through spectrum averaging. Restarting the acquisition impacts each spectrum within the electronic error of the AD detector system and defines a new calibration function. Therefore, averaging multiple independent spectra and not a larger number of laser shots leverages this inherent binning error to mitigate variability in accurate MALDI-TOF mass measurements.</p></div

Absolute values for mean, maximum and minimum errors observed for peptides from a standard protein trypsin digestion demonstrate higher variability in the more inaccurate data.

<p>Measured errors were converted to absolute values to evaluate the dispersion of data measurements and plotted according to peptide. The uneven distribution of maximum and minimum errors is expected because of zero as a lower bound for minimum error. Note, absolute value transformation of the data eliminates negative values and the mean errors reported here are higher than the mean reported for the observed peptide masses, which contain both positive and negative errors.</p

Evidence for discontinuous binning of MALDI-TOF mass spectrometry data for Des-Arg(9) Bradykinin (A), Angiotensin 1 (B), and ACTH 1–17 (C).

<p>Representative monoisotopic peaks for Des-Arg(9) Bradykinin (MH+ = 904.4676), Angiotensin 1 (MH+ = 1296.6848) and ACTH 1–17 (MH+ = 2093.0862) are enlarged to demonstrate the discontinuous sampling points (or bins) within the MALDI-TOF data. The red vertical lines are fitted to the bins evident in the observed peak shapes. The spacing of bins in mass units is larger for higher mass ions and can be accurately calculated by relationship between flight times and the ratio of masses of the molecular ions according to the equation Δt₂/Δt₁ = (M₂/M₁)^1/2. This calculation is simplistic, but accurately relates the bin spacing for different mass ions to 5 decimal places, thus suggesting that binning of data in the MALDI-TOF instrument is related to the measurements of ion flight times.</p

Identification of tubulin peptides by ProFound peptide mass fingerprinting using immunoprecipitation experiments and averaged MALDI-TOF/TOF data.

<p>Tubulin was immunoprecipitated from HEK293 cell lysate using a rabbit polyclonal antibody and Protein A/G Dynabeads. After isolation, all eluted proteins were reduced and alkylated, digested with trypsin, and analyzed on an ABI 4800 MALDI-TOF/TOF mass spectrometer. Multiple individual spectra were acquired with internal calibration and between 10 and 23 individual measurements for each peptide were used for calculating the average observed masses for each peptide. Mass Tolerance is in parts-per-million (ppm), Peptide Set defines the peptides included for search and Search Mass Range/pI Range are input parameters for Profound. Top Protein ID and Expectation Value were calculated within ProFound from the mass spectrometry data using the IPI Human database (2010-02-01).</p><p>Identification of tubulin peptides by ProFound peptide mass fingerprinting using immunoprecipitation experiments and averaged MALDI-TOF/TOF data.</p

Effect of binning and interpolation in MALDI-TOF data.

<p>The signal detected for the flight times of a packet of ions should exhibit a continuum distribution related to the energy distribution of the population of ions (TOP). However, the AD detection system can only measure ion signal from the detector in discrete time intervals represented by the red vertical lines (TOP). The discrete intensity versus time measurements are effectively bins and produce the observed discontinuous peak profile (BOTTOM). This discontinuous data must be interpolated by peak fitting algorithms to estimate the parameters of the original continuum spectrum and derive accurate mass measurements.</p

Beta-Tubulin identification and limitations of peptide mass fingerprinting.

<p>The identification of tubulin from peptide mass fingerprinting matches with LC-MS/MS data for the majority of peptides. However, assignment for the 1249.585 and 1696.805 peptides were inaccurate. The 1249.585 peak was not assigned in the MALDI-TOF data. The observed mass of 1696.805 and data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120932#pone.0120932.t003" target="_blank">Table 3</a> are from ProFound and assigned to peptide sequence ALTVSELTQQMFDSK (data denoted with an asterisk *). Peptide fragmentation data using LC-MS/MS suggests this assignment is incorrect and that the correct sequence is NSSYFVEWIPNNVK with deamidation at the amino-terminus yielding the sequence DSSYFVEWIPNNVK (MH+ = 1697.817 expected, 1697.813 observed, −2.4 ppm error).</p><p>Beta-Tubulin identification and limitations of peptide mass fingerprinting.</p

A Proteomic Signature of Dormancy in an Actinobacterium: Micrococcus luteus

This project was completed with contributions from Arshad Khan from the Department of Pathology and Laboratory Medicine, UT Health Science Center, Houston.Biology and Biochemistry, Department o

Functional and structural characterization of Hyp730, a highly conserved and dormancy‐specific hypothetical membrane protein

Abstract Membrane proteins represent major drug targets, and the ability to determine their functions, structures, and conformational changes will significantly advance mechanistic approaches to both biotechnology and bioremediation, as well as the fight against pathogenic bacteria. A pertinent example is Mycobacterium tuberculosis (H37Rv), which contains ~4000 protein‐coding genes, with almost a thousand having been categorized as ‘membrane protein’, and a few of which (~1%) have been functionally characterized and structurally modeled. However, the functions and structures of most membrane proteins that are sparsely, or only transiently, expressed, but essential in small phenotypic subpopulations or under stress conditions such as persistence or dormancy, remain unknown. Our deep quantitative proteomics profiles revealed that the hypothetical membrane protein 730 (Hyp730) WP_010079730 (protein ID Mlut_RS11895) from M. luteus is upregulated in dormancy despite a ~5‐fold reduction in overall protein diversity. Its H37Rv paralog, Rv1234, showed a similar proteomic signature, but the function of Hyp730‐like proteins has never been characterized. Here, we present an extensive proteomic and transcriptomic analysis of Hyp730 and have also characterized its in vitro recombinant expression, purification, refolding, and essentiality as well as its tertiary fold. Our biophysical studies, circular dichroism, and tryptophan fluorescence are in immediate agreement with in‐depth in silico 3D‐structure prediction, suggesting that Hyp730 is a double‐pass membrane‐spanning protein. Ablation of Hyp730‐expression did not alter M. luteus growth, indicating that Hyp730 is not essential. Structural homology comparisons showed that Hyp730 is highly conserved and non‐redundant in G+C rich Actinobacteria and might be involved, under stress conditions, in an energy‐saving role in respiration during dormancy