High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Published: August 18, 2020

doi:

Zhen Wang*¹, Kanisha Kavdia*², Kaushik Kumar Dey, Vishwajeeth Reddy Pagala, Kiran Kodali, Danting Liu, Dong Geun Lee, Huan Sun, Surendhar Reddy Chepyala, Ji-Hoon Cho, Mingming Niu, Anthony A. High, Junmin Peng²

¹Departments of Structural Biology and Developmental Neurobiology,St. Jude Children’s Research Hospital, ²Center for Proteomics and Metabolomics,St. Jude Children’s Research Hospital

Summary

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Abstract

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Introduction

Rapid developments in mass spectrometry technology have enabled to achieve high sensitivity and deep proteome coverage in proteomics applications¹^,². Despite these developments, sample multiplexing remains the bottleneck for researchers handling the analysis of a large sample cohort.

Multiplexed isobaric labeling techniques are extensively used for proteome-wide relative quantitation of large batches of samples³^,⁴^,⁵^,⁶. Tandem mass tags (TMT)-based quantitation is a popular choice for its high multiplexing capability⁷^,⁸. TMT reagents were initially launched as a 6-plex kit capable of quantifying up to 6 samples simultaneously⁹. This technology was further expanded to quantify 10-11 samples¹⁰^,¹¹. Recently developed 16-plex TMTpro (termed TMT16 hereafter) reagents have further increased the multiplexing capacity to 16 samples in a single experiment¹²^,¹³. The TMT16 reagents use a proline-based reporter group, whereas 11-plex TMT applies a dimethylpiperidine-derived reporter group. Both TMT11 and TMT16 use the same amine reactive group, but the mass balance group of TMT16 is larger than that of TMT11, enabling the combination of 8 stable C13 and N15 isotopes in the reporter ions to achieve 16 reporters (Figure 1).

The increase in multiplexing capability provides a platform for designing experiments with sufficient replicates to overcome statistical challenges¹⁴. Furthermore, the additional channels in the 16-plex TMT help reduce the total amount of starting material per channel, which may aid in the development of emerging single-cell proteomics¹⁵. The high multiplexing capacity will also be valuable in quantitation of post-translational modifications, which typically requires high amounts of starting material¹⁶^,¹⁷.

Proteomic workflows employing TMT technology have been streamlined¹⁸^,¹⁹^,²⁰, and they have evolved significantly over the past decade in terms of sample preparation, liquid chromatography separation, mass spectrometric data acquisition, and computational analysis²¹^,²²^,²³^,²⁴^,²⁵^,²⁶. Our previous article provides an in-depth overview of the 10-plex TMT platform²⁷. The protocol described here introduces a detailed, optimized method for TMT16, including protein extraction and digestion, TMT16 labeling, sample pooling and desalting, basic pH, and acidic pH reverse phase (RP) LC, high-resolution MS, and data processing (Figure 2). The protocol also highlights the key quality control steps that have been incorporated for successfully completing a quantitative proteomics experiment. This protocol can be routinely used to identify and quantify greater than 10,000 proteins with high reproducibility, to study biological pathways, cellular processes, and disease progression²⁰^,²⁸^,²⁹^,³⁰.

Protocol

Human tissues for the study were obtained with approvals from the Brain and Body Donation Program at Banner Sun Health Research Institute. 1. Protein extraction from tissue and quality control NOTE: To reduce the impact of sample harvesting on the proteome, it is crucial to collect samples in minimal time at low temperature if possible31. This is especially important when analyzing posttranslational modifications as they typically are labile, f…

Representative Results

The protocol for the newly developed TMT16, including labeling reaction, desalting, and LC-MS conditions, has been systematically optimized41. Furthermore, we directly compared the 11-plex and 16-plex methods by using them to analyze the same human AD samples41. After optimization of the key parameters for TMT16, both TMT11 and TMT16 methods yield similar proteome coverage, identification, and quantification > 100,000 peptides in > 10,000 human proteins. <p clas…

Discussion

An optimized protocol for TMT16-based deep proteome profiling has been implemented successfully in earlier publications¹²^,¹³^,⁴¹. With this current protocol, more than 10,000 unique proteins from up to 16 different samples can be routinely quantified in a single experiment with high precision.

To obtain high-quality results, it is important to pay attention to critical steps throughout the protocol. In…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was partially supported by the National Institutes of Health (R01GM114260, R01AG047928, R01AG053987, RF1AG064909, and U54NS110435) and ALSAC (American Lebanese Syrian Associated Charities). The MS analysis was performed in St. Jude Children’s Research Hospital’s Center of Proteomics and Metabolomics, which is partially supported by NIH Cancer Center Support Grant (P30CA021765). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Materials

10% Criterion TGX Precast Midi Protein Gel	Biorad	5671035
10X TGS (Tris/Glycine/SDS) Buffer	BioRad	161-0772
4–20% Criterion TGX Precast Midi Protein Gel	Biorad	5671095
50% Hydroxylamine	Thermo Scientific	90115
6 X SDS Sample Loading Buffer	Boston Bioproducts Inc	BP-111R
Ammonium Formate (NH4COOH)	Sigma	70221-25G-F
Ammonium Hydroxide, 28%	Sigma	338818-100ml
Bullet Blender	Next Advance	BB24-AU
Butterfly Portfolio Heater	Phoenix S&T	PST-BPH-20
C18 Ziptips	Harvard Apparatus	74-4607	Used for desalting
Dithiothreitol (DTT)	Sigma	D5545
DMSO	Sigma	41648
Formic Acid	Sigma	94318
Fraction Collector	Gilson	FC203B
Gel Code Blue Stain Reagent	Thermo	24592
Glass Beads	Next Advance	GB05
HEPES	Sigma	H3375
HPLC Grade Acetonitrile	Burdick & Jackson	AH015-4
HPLC Grade Water	Burdick & Jackson	AH365-4
Iodoacetamide (IAA)	Sigma	I6125
Lys-C	Wako	125-05061
Mass Spectrometer	Thermo Scientific	Q Exactive HF
MassPrep BSA Digestion Standard	Waters	186002329
Methanol	Burdick & Jackson	AH230-4
Nanoflow UPLC	Thermo Scientific	Ultimate 3000
Pierce BCA Protein Assay kit	Thermo Scientific	23225
ReproSil-Pur C18 resin, 1.9um	Dr. Maisch GmbH	r119.aq.0003
Self-Pack Columns	New Objective	PF360-75-15-N-5
SepPak 1cc 50mg	Waters	WAT054960	Used for desalting
Sodium Deoxycholate	Sigma	30970
Speedvac	Thermo Scientific	SPD11V
TMTpro 16plex Label Reagent Set	Thermo Scientific	A44520
Trifluoroacetic Acid (TFA)	Applied Biosystems	400003
Trypsin	Promega	V511C
Ultra-micro Spin Column,C18	Harvard apparatus	74-7206	Used for desalting
Urea	Sigma	U5378
Xbridge Column C18 column	Waters	186003943	Used for basic pH LC

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