The MUSC Mass Spectrometry Facility provides expertise, services, education, and state-of-the-art instrumentation to enhance biomedical research endeavors through LC-MS/MS-based proteomics. Services are offered for protein identification; characterization of post-translational modifications; and quantitative proteomics to identify differentially expressed/degraded proteins, regulated sites of post-translational modification, protein-protein interactions, and protein targets of drugs identified in phenotypic screens. Analyses include sample preparation, LC-MS/MS, database searching, generation of reports, and assistance with data interpretation. Faculty and staff assist with experimental design and the development/optimization of customized methodology for analysis of post-translationally modified peptides (e.g. phosphorylation and O-GlcNAc modification, N- and O-linked glycosylation, Cys modifications including S-glutathionylation, and glycation of Lys and Arg). Quantitative approaches including metabolic labeling (SILAC), isobaric tagging (iTRAQ/TMT), and label free proteomics (LFQ) are performed on the Orbitrap Elite or Orbitrap Fusion Lumos Mass Spectrometers. We are developing methodology to identify alterations in post-translational modifications that impact signal transduction, transcription, translation, and the response to therapeutics with the goal of enabling investigators to discover molecular mechanisms underlying disease progression and therapeutic response.

Contact Information

Lauren E. Ball, PhD, Director

(843)792-4513;

ballle@musc.edu 


Jennifer R. Bethard, MS, Facility Manager

(843)792-8637;

bethard@musc.edu


Address

Children’s Research Institute, Room 305

Medical University of South Carolina 

171 Ashley Ave., Charleston, SC 29425

Services & Pricing

MALDI-TOF MS and LC-MS/MS tandem mass spectrometry analyses are offered for protein and peptide analysis. Please contact Jennifer Bethard (bethard@musc.edu) regarding sample preparation, sample submission, and pricing. Please consult with Dr. Lauren Ball (ballle@musc.edu) regarding feasibility, experimental design, and expected results for quantitative proteomics experiments.

MALDI-tissue imaging mass spectrometry is not currently offered as a service. Please contact Dr. Richard Drake (draker@musc.edu) regarding MALDI-tissue imaging mass spectrometry.

Services Provided

Cost Estimates (MUSC Investigators)

Protein Identification (Simple or complex mixtures; in-gel or solution)
nLC-MS/MS (2 µg peptides loaded on column)$250/sample (Digestion included)
2D LC-MS/MS (MUDPIT, 5 fractions) ($125/salt step/sample)$625 (Digestion Included)
2D LC-MS/MS (MUDPIT, 10 fractions) ($125/salt step/sample)$1250 (Digestion Included)
MW Determination of Peptides or Protein by MALDI-TOF MS (Bruker)$25 (1-3 samples) $50 (4-6)
Single Protein Characterization
Characterization of Post-translational Modifications by nLC-MS/MS (Show All)

Depending on the modification(s) of interest CID, HCD, and/or ETD fragmentation is used to generate MS/MS spectra. Modified peptides are identified and the probability of site assignments are reported using Proteome Discover (SEQUEST HT and Mascot), MaxQuant (Andromeda), Protein Prospector, or Bionic. With sufficient signal (>LOQ) the relative change in the extent of modification based on peak areas of the modified and unmodified peptides (requires n≥3/condition).

$250 per LC-MS/MS analysis. (Show All)

Sites of modification are verified by manual inspection prior to site-directed mutagenesis, antibody generation, or publication. For customized database searching (unanticipated PTMs, cross-links, disulfides, labile or complex modifications), de novo sequencing, manual interpretation of spectra, and labeling of spectra $50/hr.

Phosphorylation enrichment and analysis$240/sample
De Novo Sequencing/manual interpretation of analysis$50/hr
Quantitative Proteomics for Protein Expression and Interactions
Quotes are for 3 biological replicates to comply with NIH rigor and reproducibility guidelines. The length of LC separations, number of replicates, and number of fractions can be customized.
SILAC: Stable Isotope Labeling of Amino Acids in Cell Culture (Show All)

Metabolically labeled proteins from 2 or 3 conditions are combined at equal proportions, reduced, alkylated, and digested with LysC/trypsin. Peptides (100 ug) are fractionated by high pH-RP chromatography and peptides in each fraction are analyzed by nLC-MS/MS. Proteins are identified and quantified based on relative peak intensities using MaxQuant.

$4,800 for 2 or 3-plex experiment (Show All)

with 3 biological replicates including a label swap control, each with 8 fractions (24 LC-MS/MS runs, 96hr instrument time). *Does not include cost of SILAC media or preliminary LC-MS/MS to test for >95% incorporation efficiency.

Label Free Proteomics (Show All)

Proteins from each condition (20-100 µg) are reduced, alkylated, and digested in parallel to maximize reproducibility. Samples are analyzed by nLC-MS/MS in a block-randomized format with intervening blanks to prevent carry over. Identified proteins are quantified based on relative peptide peak intensities using the MaxQuant LFQ algorithm.

$5,400 for 2 treatment groups (Show All)

each with 3 unfractionated biological replicates and 3 technical replicates (18 LC-MS/MS runs plus intervening blanks, 180hr instrument time).

Isobaric Tagging with 6-plex or 11-plex TMT or 4-plex iTRAQ (Show All)

Proteins from each condition (20-100 µg) are reduced, alkylated, digested, and labeled with amine-reactive isobaric tags that encode each of the samples in the multiplexed experiment. The samples are prepared according to the manufacturer’s instructions and the labeled peptides from each treatment group are combined at equal proportions. (This may require preliminary LC-MS/MS runs of combined aliquots to confirm equal mixing.) The combined peptides are fractionated by SCX or high pH-RP HPLC. Each fraction is analyzed by LC-MS/MS. Proteins are identified using SEQUEST HT and Mascot. Quantification is based on reporter ion intensities in the MS/MS spectrum using the Proteome Discoverer Platform. For normalization across multiple experiments, one of the labeling-reagent channels is reserved for a reference standard.

$4,350 for 12 LC-MS/MS analyses. (Show All)

*Does not include the cost of TMT or iTRAQ reagents.

Quantitative Proteomics for Post-translational Modifications
SILAC-based Phosphoproteomics (Show All)

Metabolically labeled proteins (3-5 mg per condition) are combined, reduced, alkylated, digested with LysC/trypsin and the resulting peptides are fractionated by SCX or high pH-RP HPLC. Phosphopeptides are enriched by TiO2 and analyzed by LC-MS/MS. The identification, probability of site assignment, and relative chromatographic peak intensities are extracted using MaxQuant.

$4,800 for 2 or 3-plex experiment (Show All)

with 3 biological replicates including a label swap control, each with 8 fractions (24 LC-MS/MS runs, 96 hr instrument time). *Does not include cost of SILAC media, preliminary LC-MS/MS to test for >95% incorporation efficiency, or a duplicate experiment performed with unenriched material to control for changes in protein expression.

SILAC-based Quantification of other Post-translational Modifications (Show All)

Metabolically labeled proteins are combined, reduced, alkylated, digested with LysC/trypsin and the resulting peptides may be fractionated by SCX or high pH-RP HPLC. Modified peptides are enriched by affinity purification/immunoprecipitation and analyzed by LC-MS/MS using the appropriate mode of peptide fragmentation (HCD/CID/or ETD). Depending on PTM-specific enrichment efficiency (eg acetylomics, S-glutathionylation, glycosylation…) >5 mg per condition may be required. The identification, probability of site assignment, and relative chromatographic peak intensities are extracted using MaxQuant.

Please enquire regarding feasibility, method development, and costs.
Label Free ProteomicsPlease enquire regarding feasibility and costs.
Isobaric tagging: Cys-reactive iodoTMT and amine-reactive TMT tags. (Show All)

6 or 11-plex. Amount required per condition depends on PTM enrichment efficiency, 11-plex with 100 µg sample per condition results in 1.1 mg peptides for subsequent enrichment.

Please enquire regarding feasibility, method development, and costs.
Other services
Manual inspection, de novo sequencing, and labeling of MS/MS spectra$50/hr
Off-line preparative HPLC (strong cation exchange, high pH C18 reversed phase chromatography)$50/hr
New method development/optimization/extensive data processing$50/hr or enquire regarding contributing to % effort for trained staff
Additional information
All nanoLC-MS/MS based services are currently provided using an Orbitrap Elite ETD mass spectrometer with nano-electrospray ionization. (Show All)

Identification of peptides is based on a threshold of false discovery rate (FDR) < 0.01 when searching a forward and reversed, fully tryptic protein database. Generally peptides, modified peptides, and protein groups are identified with MaxQuant using FDR thresholds of < 0.01. For global studies, class I sites of modification with probability of site localization of >75% are reported. Other search algorithms are used as needed (Protein Prospector, Byonic, Skyline, Proteome Discoverer with SEQUEST HT and Mascot). Quantification is reported with fold change and typically Student’s or Welch’s t test-derived p values adjusted for multiple hypothesis testing (q values). Statistical tests may vary with the quantitative approach. Raw data are archived off site. For publication purposes and to comply with NIH data sharing policies, proteomics data are submitted to publically available databases (ProteomeXchange) upon request.

*Prices current as of September 2018