Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap Velos.

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification; isobaric chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; and (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. On the basis of the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.

[1]  Richard J. Giannone,et al.  Defining the boundaries and characterizing the landscape of functional genome expression in vascular tissues of Populus using shotgun proteomics. , 2012, Journal of proteome research.

[2]  Jillian F. Banfield,et al.  Quantitative Tracking of Isotope Flows in Proteomes of Microbial Communities , 2011, Molecular & Cellular Proteomics.

[3]  Steven P Gygi,et al.  Proteome-wide systems analysis of a cellulosic biofuel-producing microbe , 2011, Molecular Systems Biology.

[4]  Vincent J. Denef,et al.  Quantitative proteomic analyses of the response of acidophilic microbial communities to different pH conditions , 2011, The ISME Journal.

[5]  R. Dean,et al.  Direct comparison of stable isotope labeling by amino acids in cell culture and spectral counting for quantitative proteomics. , 2010, Analytical chemistry.

[6]  Karl Mechtler,et al.  Peptide Labeling with Isobaric Tags Yields Higher Identification Rates Using iTRAQ 4-Plex Compared to TMT 6-Plex and iTRAQ 8-Plex on LTQ Orbitrap , 2010, Analytical chemistry.

[7]  N. Karp,et al.  Addressing Accuracy and Precision Issues in iTRAQ Quantitation* , 2010, Molecular & Cellular Proteomics.

[8]  Tao Xu,et al.  Bioinformatics Applications Note Sequence Analysis Xdia: Improving on the Label-free Data-independent Analysis , 2022 .

[9]  J. Koziol,et al.  Label-free, normalized quantification of complex mass spectrometry data for proteomics analysis , 2009, Nature Biotechnology.

[10]  Dorothea K. Thompson,et al.  Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge , 2010, BMC Genomics.

[11]  Matthias Mann,et al.  A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed* , 2009, Molecular & Cellular Proteomics.

[12]  Karl Mechtler,et al.  High precision quantitative proteomics using iTRAQ on an LTQ Orbitrap: a new mass spectrometric method combining the benefits of all. , 2009, Journal of proteome research.

[13]  S. Ficarro,et al.  Optimized orbitrap HCD for quantitative analysis of phosphopeptides , 2009, Journal of the American Society for Mass Spectrometry.

[14]  Konstantinos Thalassinos,et al.  A comparison of labeling and label-free mass spectrometry-based proteomics approaches. , 2009, Journal of proteome research.

[15]  Adam Godzik,et al.  Shotgun metaproteomics of the human distal gut microbiota , 2008, The ISME Journal.

[16]  Nagiza F. Samatova,et al.  Characterization of Anaerobic Catabolism of p-Coumarate in Rhodopseudomonas palustris by Integrating Transcriptomics and Quantitative Proteomics*S , 2008, Molecular & Cellular Proteomics.

[17]  D. Hochstrasser,et al.  Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. , 2008, Analytical chemistry.

[18]  Peter Lindblad,et al.  Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-plex isobaric peptide tags. , 2008, Journal of proteome research.

[19]  Timothy J Griffin,et al.  iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer. , 2007, Journal of proteome research.

[20]  Jens M. Rick,et al.  Quantitative mass spectrometry in proteomics: a critical review , 2007, Analytical and bioanalytical chemistry.

[21]  A. Chakraborty,et al.  Use of an integrated MS--multiplexed MS/MS data acquisition strategy for high-coverage peptide mapping studies. , 2007, Rapid communications in mass spectrometry : RCM.

[22]  Trong Khoa Pham,et al.  Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). , 2007, Journal of proteome research.

[23]  J. Leigh,et al.  Comparison of spectral counting and metabolic stable isotope labeling for use with quantitative microbial proteomics. , 2006, The Analyst.

[24]  N. Samatova,et al.  ProRata: A quantitative proteomics program for accurate protein abundance ratio estimation with confidence interval evaluation. , 2006, Analytical chemistry.

[25]  Michael K. Coleman,et al.  Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. , 2006, Journal of proteome research.

[26]  K. Resing,et al.  Comparison of Label-free Methods for Quantifying Human Proteins by Shotgun Proteomics*S , 2005, Molecular & Cellular Proteomics.

[27]  M. Mann,et al.  Mass spectrometry–based proteomics turns quantitative , 2005, Nature chemical biology.

[28]  Kathryn S Lilley,et al.  Impact of replicate types on proteomic expression analysis. , 2005, Journal of proteome research.

[29]  A. Heck,et al.  Double Standards in Quantitative Proteomics , 2005, Molecular & Cellular Proteomics.

[30]  K. Parker,et al.  Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents*S , 2004, Molecular & Cellular Proteomics.

[31]  John D. Venable,et al.  Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra , 2004, Nature Methods.

[32]  T. Shaler,et al.  Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. , 2003, Analytical chemistry.

[33]  Andrew H. Thompson,et al.  Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. , 2003, Analytical chemistry.

[34]  Joshua E. Elias,et al.  Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. , 2003, Journal of proteome research.

[35]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.

[36]  J. Yates,et al.  DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. , 2002, Journal of proteome research.

[37]  X. Yao,et al.  Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. , 2001, Analytical chemistry.

[38]  J. Yates,et al.  Large-scale analysis of the yeast proteome by multidimensional protein identification technology , 2001, Nature Biotechnology.

[39]  S. Gygi,et al.  Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[40]  S. Gygi,et al.  Quantitative analysis of complex protein mixtures using isotope-coded affinity tags , 1999, Nature Biotechnology.

[41]  J. Yates,et al.  An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database , 1994, Journal of the American Society for Mass Spectrometry.

[42]  P. O’Farrell High resolution two-dimensional electrophoresis of proteins. , 1975, The Journal of biological chemistry.