Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Summary

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer’s disease mouse model and wild-type controls.

Abstract

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer's disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Introduction

Proteomics often involves the analysis of many samples used to better understand disease processes, enzyme kinetics, post-translational modifications, response to environmental stimuli, response to therapeutic treatments, biomarker discovery, or drug mechanisms. Quantitative methods can be employed to measure relative differences in protein levels across the samples and can be label-free or involve isotopic labeling (metabolic, chemical, or enzymatic). Stable isotope labeling methods have grown in popularity because they allow many samples to be analyzed simultaneously and are suitable for samples from different cells, tissues, bodily fluids, or whole organisms. Isotope labeling methods^{1,2,3,4,5,6,7} increase experimental throughput, while reducing acquisition time, costs, and experimental error. These methods use precursor mass spectra to measure relative abundances of proteins from peptide peaks. In contrast, isobaric tagging reagents^8,9,10 generate reporter ions that are either detected in MS/MS or MS^{3 11} spectra and these peaks are used to report on relative abundances of proteins.

The current state-of-the-art in proteomics multiplexing is either a 10-plex¹² or 12-plex isobaric tag analysis¹³. Enhanced sample multiplexing (i.e. >10 samples) methods have been developed by our laboratory for tissues^14,15,16,17, and by others for the analysis of cells^18,19,20, tissues ²¹, or targeted peptides²². We developed an enhanced multiplexing technique called combined precursor isotopic labeling with isobaric tagging (cPILOT). Global cPILOT is useful for getting information about the relative concentrations of all proteins across different sample conditions (≥12)¹⁴. Figure 1 shows a general cPILOT workflow. Tryptic or Lys-C peptides are selectively labeled at the N-terminus with dimethylation using low pH² and at lysine residues with 6-plex reagents using high pH. This strategy doubles the number of samples that can be analyzed with isobaric reagents which helps to reduce experimental costs and additionally, reduces experimental steps and time.

cPILOT is flexible as we have developed other methods to study oxidative post-translational modifications, including 3-nitrotyrosine-modified proteins¹⁴ and cysteine containing peptides with S-nitrosylation (oxcyscPILOT)²³. We have also developed an amino acid selective approach, cysteine cPILOT (cyscPILOT)¹⁷. MS³ acquisition with a top-ion¹¹ or selective-y₁-ion method¹⁵ can help reduce reporter ion interference and improve quantitative accuracy of cPILOT. The use of MS³ in the acquisition method requires a high-resolution instrument with an orbitrap mass analyzer although low resolution ion trap instruments may also work²⁴.

Previously, cPILOT has been used to study liver proteins¹⁶ from an Alzheimer's disease mouse model. Here, we describe how to perform global cPILOT analysis using brain, heart, and liver homogenates to study the role of the periphery in Alzheimer's disease. This experiment incorporates biological replication. Because of the versatility of cPILOT, interested users can use the technique to study other tissues for a range of biological problems and systems.

Protocol

Ethics Statement: Mice were purchased from an independent, non-profit biomedical research institution and housed in the Division of Laboratory Animal Resources at the University of Pittsburgh. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. 1. Protein Extraction and Generation of Peptides for Chemical-tagging Extract protein from tissue, cells, or bodily fluids. Homogenize 60-90 mg o…

Representative Results

cPILOT uses amine-based chemistry to chemically label peptides at the N-terminus and lysine residues and enhances sample multiplexing capabilities. Figure 2 shows representative MS data that is obtained from a 12-plex cPILOT analysis of brain, heart, and liver tissues from an Alzheimer's disease mouse model and wild-type controls. As shown in Table 1, two biological replicates for the Alzheimer's disease and wild-type mice are included in this 12-…

Discussion

cPILOT allows for the simultaneous measurement of more than 12 unique samples. In order to ensure successful tagging at both the N-terminus and lysine residues of peptides, it is imperative to have the correct pH for each set of reactions and to perform the dimethylation reaction first for peptide labeling. Selective dimethylation at the N-terminus is performed by having a pH at ~2.5 (±0.2). This is achieved by exploiting the differences of the pKA's of the amino groups on lysine and the N-terminus. At pH 2.5, l…

Materials

Water – MS Grade	Fisher Scientific	W6-4	4 L quantity is not necessary
Acetonitrile – MS Grade	Fisher Scientific	A955-4	4 L quantity is not necessary
Acetic Acid	J.T. Baker	9508-01
Ammonium hydroxide solution (28 – 30%)	Sigma Aldrich	320145-500ML
Ammonium formate	Acros Organics	208-753-9
Formic Acid	Fluka Analytical	94318-250ML-F
BCA protein assay kit	Pierce Thermo Fisher Scientific	23227
Urea	Biorad	161-0731
Tris	Biorad	161-0716
Dithiothreiotol (DTT)	Fisher Scientific	BP172-5
Iodoacetamide (IAM)	Acros Organics	144-48-9
L-Cysteine	Sigma Aldrich, Chemistry	168149-25G
L-1-tosylamido-2 phenylethyl cholormethyl ketone (TPCK)-treated Trypsin from bovine pancreas	Sigma Aldrich, Life Science	T1426-100MG
Formaldehyde (CH2O) solution; 36.5 – 38% in H2O	Sigma Aldrich, Life Science	F8775-25ML
Formaldehyde (13CD2O) solution; 20 wt % in D2O, 98 atom % D, 99 atom % 13 C	Sigma Aldrich, Chemistry	596388-1G
Sodium Cyanoborohydride; reagent grade, 95%	Sigma Aldrich	156159-10G
Sodium Cyanoborodeuteride; 96 atom % D, 98% CP	Sigma Aldrich, Chemistry	190020-1G
Strong Cation Exchange (SCX) spin tips sample prep kit	Protea BioSciences	SP-155-24kit
Triethyl ammonium bicarbonate (TEAB) buffer	Sigma Aldrich, Life Science	T7408-100ML
Isobaric Tagging Kit (TMT 6 plex) – 6 reactions (1 x 0.8 mg)	Thermo Fisher Scientific	90061
Hydroxylamine hydrochloride	Sigma Aldrich, Chemistry	255580-100G
Standard vortex mixer	Fisher Scientific	2215365	any mixer can be used
Oasis HLB 1cc (10 mg)   extraction cartridges	Waters	186000383	These are C18 cartridges
Visiprep SPE vacuum manifold, DL (disposable liner), 24 port model	Sigma Aldrich	57265	A 12 port model is also sufficient
Speed-vac	Thermo Scientific	SPD1010	any brand of speed vac is sufficient
Water bath chamber	Thermo Scientific	2825/2826	Any brand of  a water bath chamber with controlled temperatures is sufficient.
Mechanical Homogenizer (i.e. FastPrep-24 5G)	MP Biomedicals	116005500
Eksigent Nano LC – Ultra 2D with Nano LC AS-2 autosampler	Sciex	–	This model is no longer available. Any nano LC with an autosampler is sufficient.
LTQ Orbitrap Velos Mass Spectrometer	Thermo Scientific	–	This model is no longer available. Other high resolution instruments (e.g. Orbitrap Elite, Orbitrap Fusion, or Orbitrap Fusion Lumos) can be used.
Protein software (e.g. Proteome Discoverer)	Thermo Scientific	IQLAAEGABSFAKJMAUH
Analytical balance	Mettler Toledo	AL54
Stir plate	VWR	12365-382	Any brand of stir plates are sufficient.
pH meter (Tris compatiable)	Fisher Scientific (Accumet)	13-620-183	Any brand of a ph meter is sufficient
pH 10 buffer	Fisher Scientific	06-664-261	Any brand of ph buffer 10 is sufficient
pH 7 buffer	Fisher Scientific	06-664-260	Any brand ph buffer 7  is sufficient
1.5 mL eppendorf tubes, 500pk	Fisher Scientific	05-408-129	Any brand of 1.5 mL eppendorf tubes are sufficient
0.6 mL eppendorf tubes, 500pk	Fisher Scientific	04-408-120	Any brand of 0.6 mL eppendorf tubes are sufficient
0.65µm Ultrafree MC DV centrifugal filter units	EMD Millipore	UFC30DV00
2 mL microcentrifuge tubes, 72 units	Thermo Scientific	69720
C18 packing material (5 µm, 100 Å)	Bruker	PM5/61100/000	This item is no longer available from Bruker. Alternative packing material with listed specifications will be sufficient.
C18 packing material (5 µm, 200 Å)	Bruker	PM5/61200/000	This item is no longer available from Bruker. Alternative packing material with listed specifications will be sufficient.