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.
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.
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 methods1,2,3,4,5,6,7 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 reagents8,9,10 generate reporter ions that are either detected in MS/MS or MS3 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 tissues14,15,16,17, and by others for the analysis of cells18,19,20, 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.
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…
The authors have nothing to disclose.
The authors acknowledge the University of Pittsburgh Start-up Funds and NIH, NIGMS R01 grant (GM 117191-01) to RASR.
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. |