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.
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.
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, shotgun proteomics approaches (Data dependent Acquisition or DDA) often fail to detect low abundant proteins in complex tissues besides being heavily reliant on the sensitivity and resolution of the instrument. Over the last couple of years, labs around the world have been developing techniques like Data Independent Acquisition (DIA) that require increased computing power and reliable software that can handle these highly complex datasets. However, these techniques are still a work in progress and not very user friendly. Targeted MS-based proteomics approaches provide a perfect balance between the high throughput nature of MS approaches and the sensitivity of molecular biology approaches like ELISA. A targeted mass spectrometry-based proteomics experiment focuses on detecting hypothesis driven proteins or peptides from discovery-based-shotgun proteomics experiments or through available literature1,2. Multiple Reaction Monitoring (MRM) is one such targeted MS approach that uses a tandem quadrupole mass spectrometer for accurate detection and quantification of proteins/peptides from complex samples. The technique offers higher sensitivity and specificity despite requiring the use of a low-resolution instrument.
A quadrupole is made of 4 parallel rods, with each rod connected to the diagonally opposite rod. A fluctuating field is created between the quadrupole rods by applying alternating RF and DC voltages. The trajectory of the ions inside the quadrupole is influenced by the presence of the same voltages across opposite rods. By applying the RF to DC voltage, the trajectory of the ions can be stabilized. It is this property of the quadrupole that allows it to be used as a mass filter which can selectively let specific ions to pass through. Depending on the need, a quadrupole can be operated in either the static mode or the scanning mode. The static mode allows only ions with a specified m/z to pass through, making the mode highly selective and specific to the ion of interest. The scanning mode on the other hand allows ions across the entire m/z range to pass through. Thus, tandem quadrupole mass spectrometers can operate in 4 possible ways: i) the first quadrupole operating in the static mode while the second operating in scanning mode; ii) the first quadrupole operating in the scanning mode while the second operating in the static mode; iii) both quadrupoles operating in the scanning mode; and iv) both quadrupoles operating in static mode3. In a typical MRM experiment, both the quadrupoles operate in the static mode allowing specific precursors and their resulting products after fragmentation to be monitored. This makes the technique very sensitive and selective allowing accurate quantification.
For molecular biologists, the human brain tissue and its cells are a treasure trove. These remarkable units of an ever-interesting organ of the human body can provide molecular and cellular insights into its functioning. Proteomic investigations of the brain tissue can not only help us understand the systemic functioning of a healthy brain but also the cellular pathways that get dysregulated when inflicted by some disease4. However, the brain tissue with all its heterogeneity is a very complex organ to analyze and requires a concerted approach for a better understanding of the changes at the molecular level. The following work describes the entire workflow starting right from extracting proteins from brain tissue, creating and optimising the methods for MRM assay, to validation of the targets (Figure 1). Here, we have systematically highlighted the major steps involved in a successful MRM based experiment using human brain tissue with an aim to introduce the technique and its challenges to a broader research community.
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
2 Protein quantification and quality check
3 Protein digestion
4 Desalting and peptide quantification
NOTE: Desalting or peptide clean-up is essential before loading the samples for LC-MS/MS. Salts and other contaminants in the sample can clog the columns and cause damage to the instrument as well. The process can be performed using commercially available C18 stage-tips or columns.
5 Transition list preparation of finalized targets
NOTE: A transition refers to the pair of precursors (Q1) to product (Q3) m/z values in an MRM experiment. A peptide can have one to many transitions, with the same Q1 value but different Q3 values. A triple quadrupole mass spectrometer requires information of the transitions for the peptides and their products to be detected. Hence, before starting a targeted experiment, a transition list needs to be prepared. This can be done using the online repository of SRMAtlas6 (https://db.systemsbiology.net/sbeams/cgi/PeptideAtlas/GetTransitions) or an open source software called Skyline7 (https://skyline.ms/project/home/software/Skyline/begin.view).
6 LC parameters
7 MS parameters
NOTE: The explained assay has been developed and optimized for TSQ Altis Triple Quadrupole Mass Spectrometer.
8 Run sequence and Instrument QC
9 Method refinement
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 and 3 peptides for NAMPT. The total number of transitions from these 10 peptides amounted to 57. The samples were grouped into either of the two groups depending on the condition they belonged to. Using the group comparisons feature of skyline, the peak abundances of these peptides were compared, and relative quantification values were calculated (Figure 3).
Figure 1: An overview of steps involved in a Multiple Reaction Monitoring experiment. A. Sample preparation for a typical proteomics experiment involves extraction of proteins (for illustration we have shown tissue sample) followed by digestion using trypsin. The digested peptides are ultimately desalted and made LC-MS ready. B. The steps involved in an MRM experiment include precursor and product ion selection based on their m/z values. Only the transitions showing good response are considered for analysis. C. The data analysis in an MRM experiment includes a detailed examination of peak shapes and peak areas. This is ultimately followed by statistical analysis of the results. Please click here to view a larger version of this figure.
Figure 2: Consistency in response for BSA using an optimized MRM method. A. Chromatogram for a representative peptide of BSA shows consistent peak shape and intensity throughout the five days the experiment was performed. B. Retention time consistency observed for the peptide on all the five days of the experiment C. Peak areas for the peptide as seen over the course of five days in the week. Please click here to view a larger version of this figure.
Figure 3: Differential regulation of three proteins in two groups of GBM tumor samples. A. Representative chromatograms for Apolipoprotein A-I and cumulative peak area as seen following inter-group comparison. B. Representative chromatograms for Vimentin and cumulative peak area as seen following inter-group comparison. C. Representative chromatograms for Nicotinamide phosphoribosyltransferase and cumulative peak area as seen following inter-group comparison. Please click here to view a larger version of this figure.
Table S1: Details of 10-minute LC gradient to be used for all samples. Please click here to download this Table.
Table S2: Parameter settings for the ion source. Please click here to download this Table.
Table S3: Parameter settings for MRM method. Please click here to download this Table.
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 using high throughput techniques like mass spectrometry, have compatible antibodies available, further complicating the whole procedure. Hence targeted proteomic assays are slowly being taken up as the new approach for validating targets10. With most of the target discovery happening on high-throughput omics platforms, panels of validated targets are also being considered for clinical screening applications11,12,13.
The representative results in this article have validated the differential expression of proteins Apolipoprotein A-I (APOA-I), Vimentin (VIM) and Nicotinamide phosphoribosyltransferase (NAMPT) in two conditions (condition 1 and condition 2) of the brain tissue. Apolipoprotein A-I has been reported to play a pivotal role in maintenance of cerebrovascular integrity and reducing the risk of Alzhiemer's disease. Even though ApoA-I is not synthesized in the brain, its ability to cross the blood brain barrier (BBB) makes its presence in the brain vital14. The Vimentin protein has been studied in a number of roles inside the brain. However, one of the key functions of Vimentin is its involvement in microglia activation. Reduced expression of Vimentin was associated with impairment of microglial activation15. The protein NAMPT has been reported to play a key role in ageing related loss of neurons and cerebral vascular endothelial dysfunctions16. All the three proteins have been reported to play a multitude of roles in normal brain cells and brain related malignancies. Therefore, MRM based validation for these proteins and their peptides can find great use in clinical diagnosis related to various brain related disorders.
A fully optimized targeted assay can be easily used for high-throughput detection and quantification of a target panel. The rate limiting step is the initial method optimization which is tedious and varies based on the sample type, protein/peptide targets, instrument being used and the detection bias of certain peptides. It is crucial that the transition list is optimized for a robust assay. Any user interested in developing such an assay for human brain tissue samples will find that the above explained protocol minimizes these variable factors. It describes an optimized protocol for peptide extraction from this unique and tedious tissue biospecimen and optimal parameters to be used in the instrument with special attention to crucial quality control steps at various points of the protocol. As with any new technology, the researchers have provided a set of guidelines, which authors need to furnish or what steps they follow during the experiment. To this front, in 2017, the MCP guidelines for reporting targeted proteomics assays and data were laid down17. These guidelines ensure that the reported study/assay is reliable and reproducible, hence increasing the applicability of the method. By taking the right precautions and utilising the true potential of this assay, researchers would soon be able to come up with clinically relevant assays with immense potential in diagnosis and therapeutics.
The authors have nothing to disclose.
We acknowledge MHRD-UAY Project (UCHHATAR AVISHKAR YOJANA), project #34_IITB to SS and MASSFIITB Facility at IIT Bombay supported by the Department of Biotechnology (BT/PR13114/INF/22/206/2015) to carry out all MS-related experiments.
We extend our special thanks to Mr. Rishabh Yadav for making and editing of the entire video and Mr. Nishant Nerurkar for his work in editing the audio.
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 |