Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Summary

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

Abstract

Recent advances in mass spectrometry have resulted in deep proteomic analysis along with the generation of robust and reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.

Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

Introduction

Proteomics technologies have the potential to enable the identification and quantification of potential candidate markers that can aid in the detection and prognostication of the disease¹. Recent advancements in the field of mass spectrometry have accelerated clinical research at the protein level. Researchers are trying to address the challenge of complicated pathobiology of several diseases using mass spectrometry-based proteomics, which now offers increased sensitivity for protein identification and quantification². Accurate quantitative measurement of proteins is crucial to comprehend the dynamic and spatial coo....

Protocol

This study was reviewed and approved by institutional review boards and the ethics committee of the Indian Institute of Technology Bombay (IITB-IEC/2016/026). The patients/participants provided their written consent to participate in this study.

1. Tissue lysate preparation

NOTE: Perform all the following steps on the ice to keep the proteases inactive. Make sure the scalpels and any tubes used are sterile to avoid any cross-contamination.

1. Take ~30 mg of tissue in a bead beating tube, add 200 µL of 1x phosphate buffer saline (PBS) and vortex it.
   NOTE: In this study, fresh ....

Results

We have used two different approaches for discovery proteomics: label-free and label-based proteomics approaches. The protein profile of tissue samples on SDS-PAGE showed the intact proteins and could be considered for proteomic analysis (Figure 2A). The quality control check of the instrument was monitored via system suitability software and it showed the day-wise variation in the instrument performance (Figure 2B). We observed 91% sequence coverage of.......

Discussion

Tissue proteomics of biological samples enables us to explore new potential biomarkers associated with different stages of disease progression. It also explains the mechanism of signaling and pathways associated with disease progression. The described protocol for tissue quantitative proteomics analysis provides reproducible good coverage data. Most of the steps have been adapted from the manufacturer's instructions. In order to obtain high-quality data, the following steps are most crucial. Hence, extra care should .......

Materials

Name	Company	Catalog Number	Comments
Reagents
Acetonitrile (MS grade)	Fisher Scientific	A/0620/21
Bovine Serum Albumin	HiMedia	TC194-25G
Calcium chloride	Fischer Scienific	BP510-500
Formic acid (MS grade)	Fisher Scientific	147930250
Iodoacetamide	Sigma	1149-25G
Isopropanol (MS grade)	Fisher Scientific	Q13827
Magnesium Chloride	Fischer Scienific	BP214-500
Methanol (MS grade)	Fisher Scientific	A456-4
MS grade water	Pierce	51140
Phosphate Buffer Saline	HiMedia	TL1006-500ML
Protease inhibitor cocktail	Roche Diagnostics	11873580001
Sodium Chloride	Merck	DF6D661300
TCEP	Sigma	646547
Tris Base	Merck	648310
Trypsin (MS grade)	Pierce	90058
Bradford Reagent	Bio-Rad	5000205
Urea	Merck	MB1D691237
Supplies
Hypersil Gold C18 column	Thermo	25002-102130
Micropipettes	Gilson	F167380
Stage tips	MilliPore	ZTC18M008
Zirconia/Silica beads	BioSpec products	11079110z
Equipment
Bead beater (Homogeniser)	Bertin Minilys	P000673-MLYS0-A
Microplate reader (spectrophotometer)	Thermo	MultiSkan Go
pH meter	Eutech	CyberScan pH 510
Probe Sonicator	Sonics Materials, Inc	VCX 130
Shaking Drybath	Thermo	88880028
Orbitrap Fusion mass spectrometer	Thermo	FSN 10452
Nano LC	Thermo	EASY-nLC1200
Vacuum concentrator	Thermo	Savant ISS 110
Software
Proteome Discoverer	Thrermo	Proteome Discoverer 2.2.0.388