Global relative and absolute quantitation in microbial proteomics

Otto, Andreas; Bernhardt, Jörg; Hecker, Michael; Becher, Dörte

doi:10.1016/j.mib.2012.02.005

Cited by 39 publications

(25 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The protein concentration was determined with the Roti-Nanoquant protein assay (Roth, Karlsruhe, Germany) according to the manufacturer's instructions, and the same protein amount of 15 N-labeled standard was added. The sample was analyzed using the GeLC-MS workflow described by Otto et al (5). The proteins were separated via one-dimensional SDS-PAGE; the gel was cut into 12 pieces, which were tryptically digested at 37°C overnight.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Heßling

Büttner

Hecker

et al. 2013

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LCelectrospray ionization workflows.Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis.Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF-and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments. One-dimensional gel-based liquid chromatography mass spectrometry (GeLC-MS) 1 is a well-established technique in life science. In combination with in vivo labeling approaches such as stable isotope labeling by amino acids in cell culture (SILAC) (1) or 15 N labeling (2), it allows the relative quantification of large numbers of proteins in a complex sample. Mass spectrometry (MS) measurements in such workflows are predominantly performed with electrospray ionization (ESI)-based mass spectrometers. Online coupling of a liquid chromatography (LC) system with fast MS spectra acquisition and high-mass-accuracy ESI instruments in conjunction with fractionation on both protein and peptide levels allows the analysis of very complex samples in a relatively short period of time (3). The identification and quantification of data can be done in an automatic or semi-automatic manner with a variety of well-established software packages (4).In proteomic research, matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF) is mainly used for the analysis of non-complex protein samples that do not require fractionation on the peptide level by means of liquid chromatography. In particular, the analysis of single protein spots resulting from the twodimensional PAGE separation of complex samples is primarily carried out with this MS tec...

show abstract

Section: Methodsmentioning

confidence: 99%

“…In particular, the analysis of single protein spots resulting from the twodimensional PAGE separation of complex samples is primarily carried out with this MS technique, as it allows the fast and reliable analysis of a high number of low-complex samples (5).…”

mentioning

confidence: 99%

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Heßling

Büttner

Hecker

et al. 2013

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

show abstract

“…To emphasize highly expressed proteins, each protein is associated with a polygon-shaped tile whose size is proportional to that protein's abundance. Although treemaps have already been used to encode expression changes by colors (23)(24)(25), we encode protein abundance directly by size. We display mass fractions: i.e., protein copy numbers weighted by the chain lengths, thus showing the amino acid investment for protein production and maintenance.…”

mentioning

confidence: 99%

Visual account of protein investment in cellular functions

Liebermeister

Noor

Flamholz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

330

295

View full text Add to dashboard Cite

Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held desire to measure absolute protein abundances on a genome-wide scale. However, can this knowledge be translated into a clearer picture of how cells invest their protein resources? This article aims to give a broad perspective on the composition of proteomes as gleaned from recent quantitative proteomics studies. We describe proteomaps, an approach for visualizing the composition of proteomes with a focus on protein abundances and functions. In proteomaps, each protein is shown as a polygon-shaped tile, with an area representing protein abundance. Functionally related proteins appear in adjacent regions. General trends in proteomes, such as the dominance of metabolism and protein production, become easily visible. We make interactive visualizations of published proteome datasets accessible at www.proteomaps.net. We suggest that evaluating the way protein resources are allocated by various organisms and cell types in different conditions will sharpen our understanding of how and why cells regulate the composition of their proteomes.Voronoi treemap | functional classification | mass spectrometry | cell resource allocation | cellular economy

show abstract

“…For the 'discovery driven' approach, several proteomic methodologies are available, they can be distinguished as the gel-based and the gel-free methods and excellent reviews recently discussed technological advances in the microbial proteomics (Armengaud 2013;Gil and Monteoliva 2014;Otto et al 2014Otto et al , 2012Oudenhove and Devreese 2013). Briefly, the gel-based approaches are based on protein separation by the two-dimensional (2D) gel electrophoresis followed by protein spot isolation and identification by mass spectrometry, while the gel-free proteomics is based on the enzymatic digestion of the protein mixture followed by liquid chromatography and identification of peptide sequences by mass spectrometry (Otto et al 2012;Wöhlbrand et al 2013). Although the greater part of biocontrol proteomics studies were carried out using the more traditional gel-based methods (Table 3), these approaches are subjected to the limitation of scarce reproducibility and limited numbers of samples and proteins that can be analyzed for each experiment.…”

Section: Proteomic Analyses Of Biocontrol Agentsmentioning

confidence: 99%

Impact of the omic technologies for understanding the modes of action of biological control agents against plant pathogens

et al. 2015

View full text Add to dashboard Cite

The characterization of microbial biological control agents (MBCAs) is crucial to improve their efficacy and consistency as biopesticides. Powerful approaches to characterize MBCA's modes of action are provided by modern molecular technologies. This paper reviews improvements achieved in this subject by three ''omics'' approaches: namely the genomic, the transcriptomic and the proteomic approaches. The paper discusses the advantages and drawbacks of new molecular techniques and 'discovery driven' approaches to the study of the biocontrol properties against plant pathogens. Omics technologies are capable of: (i) identifying the genome, transcriptome or proteome features of an MBCA strain, (ii) comparing properties of strains/mutants with different biocontrol efficacy, (iii) identifying and characterizing genes, mRNAs and proteins involved in MBCA modes of action, and (iv) simultaneously studying the transcriptome or proteome of the plant host, the plant pathogen and the MBCAs in relation to their bi-or tri-trophic interactions.

show abstract

Global relative and absolute quantitation in microbial proteomics

Cited by 39 publications

References 57 publications

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Visual account of protein investment in cellular functions

Impact of the omic technologies for understanding the modes of action of biological control agents against plant pathogens

Contact Info

Product

Resources

About