Director, Robert N. Cole, Ph.D.
Associate Director, Sandra Gabelli, Ph.D.
Proteomics Specialist, Tatiana N. Boronina, Ph.D.
Bioinformatician, C. Conover Talbot
The Proteomics Core applies cutting-edge mass spectrometry technology to identify, quantify and characterize proteins or their modifications for assisting Center Members in elucidating the molecular mechanisms mediating normal and pathophysiology in the small intestine, colon, liver/biliary and pancreas.
The Proteomics Core (Core Director and Proteomics Specialist) offers consultation on strategies for experimental design, protein extraction, and post-analysis data interpretation, and assists in proteomic data repository submission, as stated in the Operational Plan, at no charge to the JHU DDRCC Member.
Proteomics Core staff provide the following sample preparation services: buffer exchange (TCA/acetone precipitation, affinity or filtration spin columns, affinity resin tip, or plate desalting); reduction and alkylation of proteins; proteolytic digestions; isobaric mass tag labeling (TMTs); phosphopeptide enrichment (TiO2) or advice on immunoaffinity enrichment for modified peptides; and peptide fractionation by basic reverse phase (bRP) chromatography. The Proteomics Specialist also trains JHU DDRCC Investigators interested in performing these techniques and oversees or trains students and fellows in current protein extraction and sample preparation techniques to ensure the quality, reproducibility, and continuity of sample preparation.
The Proteomics Core routinely identifies and characterizes proteins from complex protein extracts, or in gel bands or spots, by nanoflow liquid chromatography/tandem mass spectrometry (nanoLCMS/MS) using the single or multi-dimensional protein identification technology (MuDPIT). Our standard nanoLCMS/MS analysis is performed on a 75 μm × 150 mm reverse-phase (ProntoSIL-120-5-C18 H column, 5 µm, 120Å (BISCHOFF)) column using 2%-90% acetonitrile/0.1% FA gradient over 90 min at 300 nL/min on a Thermo Easy-nLC 1000 or 1200 interfaced with our Thermo Orbitraps. Eluting peptides are sprayed into the mass spectrometer through a 1 µm emitter tip (New Objective) at 2.4 kV. Survey scans (full ms) are acquired on the Orbitrap within 350-1800 m/z using the data-dependent top 15 method with dynamic exclusion of 15 s. Precursor ions are individually isolated with 0.7 m/z, and fragmented using HCD activation collision energy 28. Precursor and product ions are analyzed at resolution 120,000/ >30,000 at 200 m/z, AGC targets 3xe6/1xe5, max IT 60 ms/150 ms, respectively, in FTFT mode. Proteome Discoverer (PD) extracts and searches MS data against protein databases using the Proteomics Core’s three Mascot licenses to identify proteins in Center Investigator samples. Mascot search results are imported into Scaffold for reporting results to the JHU DDRCC Investigator. The Proteomics Specialist reviews Scaffold results with Investigators to assist with determining significant protein identifications based on the standard criteria of > 2 unique peptides identified from the same protein with individual peptide confidence probability scores >95% (<5% FDR) and sub 3 ppm mass error on precursor and productions. JHU DDRCC Investigators receive an interactive Scaffold file containing annotated data supporting all proteins identified.
JHU DDRCC Members used the Proteomics Core to identify and map acetylation, biotinylation, O-GlcNAcylation, lipidation, oxidation, phosphorylation, sulfation, and ubiquitination. Proteomics Core staff performed or trained JHU DDRCC Investigators to enrich samples for modified peptides using TiO2 for Ser/Thr/ Tyr phosphorylated peptides. Immunoaffinity enrichment of Tyr phosphorylated, acetylated, or ubiquitinated peptides is performed in the JHU DDRCC Member’s lab using antibodies from Cell Signaling under the guidance of the Proteomics Specialist.
The Proteomics Core primarily performs relative quantitative proteomics using isobaric mass tags, Tandem Mass Tags (TMTs, Thermo Fisher Scientific), in combination with MuDPIT using standalone (off-line) basic reverse phase (bRP) chromatographyfollowed by acidic reverse phase nanoLCMS/MS as described above. The analysis includes experimental design to compare up to 16 samples in a single TMT experiment, protein digestions, TMT labeling, bRP separation and concatenation into 24 fractions, nanoLCMS/MS of each fraction, and protein identification and quantification. Fractionating the combined TMT labeled peptides in 24 fractions significantly reduces co-isolation of multiple peptides, reduces report ion suppression, and increases quantification accuracy. Investigators receive an interactive PD file containing quantification of all proteins and modified peptides identified, and from which they can create heat maps and principal component analysis (PCA) and volcano plots.
The Proteomics Core combines the above relative protein quantification method with enrichment strategies for modified peptides to quantify PTMs. These workflows start with at least 0.1 mg of total protein per sample. After TMT labeling, 5% of the combined peptide labeled samples is analyzed before enrichment for relative protein quantification. The remaining 95% is analyzed after enrichment for labeled PTM modified peptides. With this approach, thousands of modified peptides have been identified and quantified for JHU DDRCC Investigators. Changes in the abundance of a modified site due to site occupancy are distinguished from changes in protein abundances, if the time course of the experiment precludes changes due to protein synthesis, or if the modified proteins are also identified before enrichment and their abundance did not change. The Core has quantified oxidation, PEGylation, and phosphorylation sites for JHU DDRCC members, but is capable of quantifying other PTMs if requested.
Data Analysis and Bioinformatics
The Proteomics Core has several bioinformatics programs to identify, quantify, and characterize proteins. Free viewers of all bioinformatics software are available to JHU DDRCC Members. They can use the viewer software or work with the Proteomics Core bioinformatician to analyze and visualize their data using PCA, volcano plots, or heat maps to identify proteins of interest and map proteomic results to canonical pathways using Ingenuity software. The Proteomics Specialist will transfer data to and works with the bioinformatician on post-data acquisition analyses, and assists investigators with uploading MS data and results to public proteomic data repositories.
One-on-one training on sample preparation and data analysis by the Proteomics Specialist is encouraged and is available to all JHU DDRCC Member labs at no charge. Training and discussions of advantages, limitations and alternatives of current protein extraction and sample preparation techniques; quality controls and procedures, such as SDS-PAGE as a simple cost-effective control for sample preparation reproducibility. Users are informed about standard operating procedures (SOPs) for preparing and submitting gel bands or protein extracts for protein identification or relative quantification; and assisted with interpretation of MS spectra for protein identification or PTM mapping (emphasis on recognizing diagnostic fragment ions); and statistical criteria for significant changes in protein or modification abundances. Providing instruction in these areas ensures the quality, reproducibility, and uniformity of sample preparation for high-quality data acquisition and data interpretation.
Changes in Services
The Proteomics Core continues to develop new services in response to the research needs of JHU DDRCC Members. New methods are developed either in the Core or in collaboration with Investigators and are based on advances in proteomics and Investigator requests. When ready for implementation, the new services are presented for approval by the Proteomics Core Director to the MSPF’s Scientific Advisory Board. JHU DDRCC Members are made aware of new services by Work-in-Progress seminars and at the annual JHU DDRCC Research Base retreat. The decision to continue, add, or remove Proteomics Core services is based on JHU DDRCC Member feedback from informal consultations, the Work-in-Progress meetings, the yearly Research Base retreat and survey, and yearly discussions with the JHU DDRCC External Scientific Advisory Committee. Those services with low JHU DDRCC Member usage are considered for removal.
(a) Services deleted: Due to low usage, mass measurement by MALDI-TOF analysis, high-resolution mass spectrometry (HRMS) analysis, and MALDI-TOF and 2D gel electrophoresis training are no longer offered as services. The MALDI-TOF and 2D gel electrophoresis equipment are still available for self-use if needed, after training by Proteomics Core staff. The Proteomics Core also no longer offers quantitative proteomics using the iTRAQ 8-plex isobaric mass tags because the iTRAQ reagents have been replaced by the next generation isobaric mass tags, TMTs, with TMT 11-plex and TMTpro 16-plex tags.
(b) Services added: In response to requests from JHU DDRCC Members, the Proteomics Core now offers a higher TMT multiplex version of TMTs using TMTpro 16-plex reagents and two new sample preparation services for PTM preservation using a Denator and protein extraction using a Barocycler.
1) Multiplex Protein Quantification for 16 Samples per Experiment: The Proteomics Core can now compare protein abundances in up to 16 samples in a single TMT quantitative experiment. The TMTpro 16-plex isobaric mass tag reagents became commercially available from Thermo Fisher Scientific in September 2019. The labeling chemistry and workflows using TMTpro 16plex reagents are the same as for the TMT 11-plex reagents. The TMTpro 16-plex reagents are not isobaric with the TMT 11-plex reagents. Both TMT versions are available to JHU DDRCC members. The new 16 plex has been used by Lutsenko and Donowitz laboratories.
2) PTM Preservation in Tissues: In response to JHU DDRCC member needs to detect low abundance or small changes in phosphorylation or O-GlcNAcylation modifications, a heat stabilizer unit called the Denator (purchased in part with JHU DDRCC funds) was made available to JHU DDRCC Members to preserve these PTMs. The Denator uses rapid conductive heating of small tissue sections for fast protein denaturation to inhibit all enzymatic activity. The Denator is housed and managed by JHU DDRCC Member Brian O’Rourke’s lab in collaboration with the Proteomics Core and is being used by the O’Rourke, Donowitz and V. Singh laboratories.
3) Protein Extraction and Digestion by Pressure Cycling: To reduce sample handling time and streamline protein extraction, the Core added pressure-cycling technology to the sample preparation services. Proteolytic digestion times are reduced to under an hour by pressure-cycling in a Barocycler 2320 Extreme (Pressure Biosciences). However, pressure cycling will also lyse cells. To obtain tryptic peptides from cells or organoids in a single step, the Proteomics Core combines cell or organoid lysis and proteolytic digestion in the Barocycler. Thus, reducing sample processing time and handling, and reducing the number of cells required for protein analysis. The Proteomics Core has used this application with TMT labeling to compare protein profiles between normal and Loeys-Dietz esophageal organoids for the Guerrerio Lab and normal versus knockout esophageal organoids for the Meltzer Lab studying Barrett’s disease. Using <200 organoids, >5000 protein groups were quantified with remarkable reproducibility (<10%CVs) in label-free analysis.
SERVICES AND FEES
|Service||Includes||Unit||DDRCC Unit Cost*||Non-Member Unit Cost|
|Consultation||Experimental Design, Sample Preparation, Data Analysis, Data Repositories||per project||no charge||no charge|
|Sample Preparation||Buffer Exchange||per sample||$20||$40|
|Proteolys is (in gel or solution)||per sample||$28||$55|
|Proteolysis (FASP)||per sample||$40||$80|
|Phosphopeptide Enrichment (T102)||per sample||$20||$40|
|Mass Tag Labeling (TMT)||per sample||$75||$150|
|Fractionation (5 step gradient)||per step||$10||$20|
|Fractionation (linear gradient)||per gradient||$400||$880|
|Protein Identification||nLCMS/MS only||per fraction||$60||$120|
|Buffer Exchange, Proteolysis,|
nLCMS/MS and Data analysis, no
Protein Identification fractionation
|MuDPIT Analysis (Buffer Exchange,|
Proteolys is 24 Fractions, nLCMS/MS,
|Protein Modification Identification||Phosphopeptide Analysis (Buffer Exchange, Proteolysis, TiO2|
Identification Enrichment, nLCMS/MS (before and
after enrichment), Data Analysis
|Protein Quantification (isobaric mass tags)||TMT 11 plex with MuDPIT Analysis||per 11 samples||$3,310||$6,620|
|TMT 16 plex with MuDPIT Analysis||per 16 samples||$3,923||$7,845|
|Protein Modification Quantification (isobaric mass tags)||TMT 11 plex with MuDPIT Analysis (24 fractions before and 12 fractions after enrichment)||per 11 samples||$4,430||$8,860|
|TMT 16 plex with MuDPIT Analysis (24 fractions before and 12 fractions after enrichment)||per 16 samples||$4,943||$9,885|
|Data Analysis and|
|Database Search||per fraction||$5||$10|
|Custom Database||per database||$28||$55|
|PCA Volcano Plots, Heat Maps in|
|per project||no charge||no charge|
|Ingenuity Pathway Analysis||per hour||$100||$200|
|Training||Scaffold, PEAKS, Byonic, Proteome Discoverer, Skyline||per project||no charge||no charge|
*JHU DDRCC Full and Associate member costs are offset ~50% and 75%, respectively, from non-center and Collaborating Member rates. Shown are Full Member rates. Associate Members pay half the Full Member rates.