A Proteomics Search Algorithm Specifically Designed for High-Resolution Tandem Mass Spectra

The acquisition of high-resolution tandem mass spectra (MS/MS) is becoming more prevalent in proteomics, but most researchers employ peptide identification algorithms that were designed prior to this development. Here, we demonstrate new software, Morpheus, designed specifically for high–mass accuracy data, based on a simple score that is little more than the number of matching products. For a diverse collection of data sets from a variety of organisms (E. coli, yeast, human) acquired on a variety of instruments (quadrupole–time-of-flight, ion trap–orbitrap, and quadrupole–orbitrap) in different laboratories, Morpheus gives more spectrum, peptide, and protein identifications at a 1% false discovery rate (FDR) than Mascot, Open Mass Spectrometry Search Algorithm (OMSSA), and Sequest. Additionally, Morpheus is 1.5 to 4.6 times faster, depending on the data set, than the next fastest algorithm, OMSSA. Morpheus was developed in C# .NET and is available free and open source under a permissive license

Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics

Large-scale protein sequencing methods rely on enzymatic digestion of complex protein mixtures to generate a collection of peptides for mass spectrometric analysis. Here we examine the use of multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) to improve both protein identification and characterization in the model organism Saccharomyces cerevisiae. Using a data-dependent, decision tree-based algorithm to tailor MS2 fragmentation method to peptide precursor, we identified 92 095 unique peptides (609 665 total) mapping to 3908 proteins at a 1% false discovery rate (FDR). These results were a significant improvement upon data from a single protease digest (trypsin) − 27 822 unique peptides corresponding to 3313 proteins. The additional 595 protein identifications were mainly from those at low abundances (i.e., < 1000 copies/cell); sequence coverage for these proteins was likewise improved nearly 3-fold. We demonstrate that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than technical replicates, provide a direct route to increase both protein identifications and proteome sequence coverage

Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics

Large-scale protein sequencing methods rely on enzymatic digestion of complex protein mixtures to generate a collection of peptides for mass spectrometric analysis. Here we examine the use of multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) to improve both protein identification and characterization in the model organism Saccharomyces cerevisiae. Using a data-dependent, decision tree-based algorithm to tailor MS2 fragmentation method to peptide precursor, we identified 92 095 unique peptides (609 665 total) mapping to 3908 proteins at a 1% false discovery rate (FDR). These results were a significant improvement upon data from a single protease digest (trypsin) − 27 822 unique peptides corresponding to 3313 proteins. The additional 595 protein identifications were mainly from those at low abundances (i.e., < 1000 copies/cell); sequence coverage for these proteins was likewise improved nearly 3-fold. We demonstrate that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than technical replicates, provide a direct route to increase both protein identifications and proteome sequence coverage

Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics

Large-scale protein sequencing methods rely on enzymatic digestion of complex protein mixtures to generate a collection of peptides for mass spectrometric analysis. Here we examine the use of multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) to improve both protein identification and characterization in the model organism Saccharomyces cerevisiae. Using a data-dependent, decision tree-based algorithm to tailor MS2 fragmentation method to peptide precursor, we identified 92 095 unique peptides (609 665 total) mapping to 3908 proteins at a 1% false discovery rate (FDR). These results were a significant improvement upon data from a single protease digest (trypsin) − 27 822 unique peptides corresponding to 3313 proteins. The additional 595 protein identifications were mainly from those at low abundances (i.e., < 1000 copies/cell); sequence coverage for these proteins was likewise improved nearly 3-fold. We demonstrate that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than technical replicates, provide a direct route to increase both protein identifications and proteome sequence coverage

Image_1_Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions.TIFF

Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.</p

Organic Acid Quantitation by NeuCode Methylamidation

We have developed a multiplexed quantitative analysis method for carboxylic acids by liquid chromatography high resolution mass spectrometry. The method employs neutron encoded (NeuCode) methylamine labels (¹³C or ¹⁵N enriched) that are affixed to carboxylic acid functional groups to enable duplex quantitation via mass defect measurement. This work presents the first application of NeuCode quantitation to small molecules. We have applied this technique to detect adulteration of olive oil by quantitative analysis of fatty acid methyl amide derivatives, and the quantitative accuracy of the NeuCode analysis was validated by GC/MS. Currently, the method enables duplex quantitation and is expandable to at least 6-plex analysis

<i>isp2Δ</i> spores show a delay in germination.

<p>(A) <i>isp2Δ</i> spores have a germination defect on solid YPD medium. Colonies of wild type and <i>isp2Δ</i> strains after growth at room temperature for 63h (germination) and 51h (vegetative growth). Scale bars, 100μm (5x magnification). (B) Quantification of colony sizes using ImageJ. The average colony size of <i>isp2Δ</i> spores was only 20.5% of wild type spores, whereas the average colony size of <i>isp2Δ</i> yeast was 45.7% of wild type yeast. The difference in size between colonies from yeast growth and spore germination for <i>isp2Δ</i> strains was significant (p = 7.1x10^-48) but not for the wild type strain (p = 7.8x10^-1). Data represent number (n) of independent experiments and are shown as a mean ± SD. An unpaired two-sided Student's t-test was used to assess significance. (C) Germination delay for <i>isp2Δ</i> spores in liquid YPD media. Optical density at a wavelength of 600nm (OD₆₀₀) was measured every 3min over 50h. The y-axis shows OD₆₀₀ and the x-axis shows time in hours (h). Plots are representative of three independent experiments. (D) Average time taken to double initial OD₆₀₀. Quantified doubling times were nearly identical for wild type and <i>isp2Δ</i> yeast (p = 0.38); however, <i>isp2Δ</i> spores took significantly longer than wild type to double the population (p = 1.2×10⁻¹⁰). Data represent number (n) of independent experiments and are shown as mean ± SD. An unpaired two-sided Student's t-test was used to assess significance. (E) Morphological changes during germination of <i>isp2Δ</i> and wild type spores. Spores were exposed to YPD liquid media to trigger germination at room temperature and photographed at 0h and 12h. Scale bars, 5μm (1000× magnification).</p

Table_4_Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions.XLSX

Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.</p

Summary of spore-enriched protein mutant phenotypes.

<p><i>C</i>. <i>neoformans</i> spores are produced through sexual development (above the gray bar). Sexual development occurs between haploid yeast of opposite mating types (<b>a</b> and α) and includes mate detection and cell fusion, dikaryotic filamentation, basidium formation and production of spores in chains. Spores can disperse and germinate into yeast (below the gray bar) to complete the life cycle. Gene names in red text are placed at the process in which deletion mutants show phenotypes. Six genes, <i>ISP3-7</i> and <i>SFH5</i>, did not result in detectable phenotypes in any assays.</p