Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

Summary

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

Abstract

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

Introduction

During the last decade, rapid developments in mass spectrometry (MS) coupled with increased understanding of chromatography techniques have greatly helped in advancement of MS-based proteomics. Molecular biology-based techniques such as western blotting and immunohistochemistry have long suffered from reproducibility issues, slow turnaround time, inter-observer variability and their inability to accurately quantify proteins, to name a few. To this end, the superior sensitivity of high-throughput proteomics approaches continues to offer molecular biologists an alternate and more reliable tool in their quest to better understand the roles of proteins in cells. However, ....

Protocol

This study involves brain tissue samples from human participants, reviewed and approved by TMH and IITB IEC - (IITB-IEC/2018/019). The participants provided their informed and written consent to participate in this study.

1 Protein extraction from brain tissue

1. Weigh around 50 mg of brain tissue and wash the tissue with 300 µL of 1x phosphate buffer saline (PBS) using a micropipette.
   NOTE: This step is performed to remove any blood on the external surface of the tissue and must be repeated if necessary. It is advisable to remove as much blood from tissue as possible as it interferes with downstream protein estima....

Results

We performed relative quantification of 3 proteins from 10 samples, 5 samples from each group of patients with abnormalities in the brain. These proteins included Apolipoprotein A-I (APOA-I), Vimentin (VIM) and Nicotinamide phosphoribosyltransferase (NAMPT) which are known to perform diverse roles in the brain cells. Post-run analysis of the data was performed using Skyline-daily (Ver 20.2.1.286). A total of 10 peptides corresponding to 3 proteins were monitored. These included 3 peptides for APOA-I, 4 peptides for VIM a.......

Discussion

Techniques like Immunohistochemistry and Western blotting were considered as the gold standards for validation of protein targets for many years. These methods find use even today with minor modifications in the protocol and little dependence on technology making them very cumbersome and tedious. Besides this, they also involve the use of expensive antibodies which do not always show the same specificity across batches and require a great deal of expertise. Additionally, only a small fraction of proteins identified .......

Materials

List of materials used in this article

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
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
TSQ Altis mass spectrometer	Thermo	TSQ02-10002
uHPLC - Vanquish	Thermo	VQF01-20001
Vacuum concentrator	Thermo	Savant ISS 110