Method Article

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

DOI:

10.3791/56474

⸱

November 15th, 2017

 ,  ,  ,  ,  ,  ,  , 
1St. Jude Proteomics Facility, St. Jude Children's Research Hospital, 2Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital, 3Heilongjiang University of Chinese Medicine

In This Article

Summary

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We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Abstract

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Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

Introduction

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Advances in next-generation sequencing technology have led to a new landscape for studying biological systems and human disease. This has permitted a large number of measurements of the genome, transcriptome, proteome, metabolome, and other molecular systems to become tangible. Mass spectrometry (MS) is one of the most sensitive methods in analytical chemistry, and its application in proteomics has rapidly expanded after the sequencing of the human genome. In the proteomics field, the past few years have yielded major technical advances in MS-based quantitative analyses, including isobaric labeling and multiplexing capability combined with extensive liquid chromatogra....

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Protocol

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CAUTION: Please consult all relevant safety data sheets (i.e., MSDS) before use. Please use all appropriate safety practices when performing this protocol.

NOTE: A TMT 10-plex isobaric label reagent set is used in this protocol for the proteome quantitation of 10 samples.

1. Preparation of Cells/Tissues

NOTE: It is critical to collect samples in minimal time at low temperature to keep proteins in their original biological state.

  1. Wash adherent cells (e.g., HEK 293 cells) on a 10 cm plate twice with 10 mL of ice cold phosphate-buffered saline (PBS). Scrape a....

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Results

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We used a previously described cross-species peptide mix to systematically analyze the effect of ratio compression in 3 major protocol steps, including pre-MS fractionation, MS settings, and post-MS correction23. The pre-MS fractionation was evaluated and optimized by using a combination of basic pH RPLC and acidic pH RPLC. For post-MS analysis, only species-specific peptides were considered. We used this interference model to examine a number of parameters in LC/L.......

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Discussion

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We describe a high-throughput protocol for the quantitation of proteins with a 10-plex isobaric labeling strategy, which has been implemented successfully in several publications12,13,14,32. In this protocol, we can analyze up to 10 different biological protein samples in 1 experiment. We can routinely identify and quantitate well over 10,000 proteins with high confidence. Although isobaric lab.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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The authors thank all other lab and facility members for helpful discussion. This work was partially supported by NI H grants R01GM114260, R01AG047928, R01AG053987, and ALSAC. The MS analysis was performed in the St. Jude Children's Research Hospital Proteomics Facility, partially supported by NIH Cancer Center Support grant P30CA021765. The authors thank Nisha Badders for help with editing the manuscript.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1220 LC systemAgilentG4288B
50% HydroxylamineThermo Scientific90115
AcetonitrileBurdick & JacksonAH015-4
Bullet BlenderNext AdvanceBB24-AU
Butterfly Portfolio HeaterPhoenix S&TPST-BPH-20
C18 tipsHarvard Apparatus74-4607
Dithiothreitol (DTT)SigmaD5545
DMSOSigma41648
Formic acidSigma94318
Fraction CollectorGilsonFC203B
Glass BeadsNext AdvanceGB05
HEPESSigmaH3375
Iodoacetamide (IAA)SigmaI6125
Lys-CWako125-05061
MethanolBurdick & JacksonAH230-4
Pierce BCA Protein Assay kitThermo Scientific23225
Mass SpectrometerThermo ScientificQ Exactive HF
nanoflow UPLCThermo ScientificUltimate 3000
ReproSil-Pur C18 resin, 1.9umDr. Maisch GmbHr119.aq.0003
Self Pck ColumnsNew ObjectivePF360-75-15-N-5
Sodium deoxycholateSigma30970
SpeedvaThermo ScientificSPD11V
TMT 10plex Isobaric label reagentThermo Scientific90110
Trifluoroacetic acid (TFA)Applied Biosystems400003
TrypsinPromegaV511C
UreaSigmaU5378
Xbridge Column C18 columnWaters186003943
Ziptips C18MilliporeZTC18S096
SepPak 1cc 50mgWatersWAT054960

References

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  1. Pagala, V. R., et al. Quantitative protein analysis by mass spectrometry. Methods Mol Biol. 1278, 281-305 (2015).
  2. Altelaar, A. F., Munoz, J., Heck, A. J. Next-generation proteomics: towards an integrative view of proteome dynamics. Nat Rev Genet. 14

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Tags

Deep Proteome ProfilingIsobaric LabelingLiquid ChromatographyMass SpectrometryTMT Labeling2D LCHigh Resolution Mass SpectrometryComputational Data ProcessingProtein QuantitationPost Translational Modifications

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