Proteomics and Metabolomics

Proteomics and Metabolomics
PROTEOMICS AND METABOLOMICS

Services


The Proteomics and Metabolomics Facility (PMF) at the Center for Biotechnology, Beadle Center offers a range of technical services using mass spectrometry, including identification and relative quantification of proteins and several advanced methods for profiling and quantitation of small molecules. With highly specialized technology capabilities and proficiency and expertise of personnel in the field of mass spectrometry, the facility is well-prepared and positioned to serve as a regional, national, and international resource and to foster collaboration in the fast developing field of proteomics and metabolomics.

Please send an email if you have any questions regarding these services and/or their costs. The email address to submit samples to can be found in the Sample Submission Form, which needs to be enclosed at submission with the samples and also sent electronically. Scientists must contact us before submitting samples to check for compatibility of sample type and format with our analytical approaches and to confirm the experimental approach and expectations for the services.

Proteomics services include:

  • Bottom-up Proteomics of whole organisms, organs, organelles, tissues, cell pellets, pulldowns, complex or single protein mixes from solutions or gels.
  • Quantification by label-free or multiplexed labeling techniques (eg TMT-10/16plex) with microscale subfractionation to increase coverage.
  • Post Translational Modification Characterization especially phosphopeptide enrichment from whole organism digests, but also ubiquitylation, acetylation, etc.

Metabolomics services include:

  • Targeted Quantification using HPLC or LCMS. See table below for a full list of compounds.

Targeted Primary Metabolites
Polyamines Putrescine, Spermine, Spermidine
Pyrimidine Pathway    ATP, GTP, dATP, dGTP, dCTP, UTP, dUTP, Uridine, UMP, UDP, dCDP, CTP, CDP, Cytidine, dUMP, Deoxyuridine, dTDP, Thymidine, dTTP, car-Asp, dTMP
Free Sugars Fructose, Sucrose, Glucose, Raffinose, Arabinose, Mannose, Xylose, Galactose, Lactose, Pinitol, Trehalose
Free Amino Acids 20 Amino Acids, Tau, GABA, Orn, HyPro
Hydrolyzed Amino Acids A, I, L, V, F, Y, D, E, MetS, CyA, S, T, R, H, K, G, P
TCA Cycle /Glycolysis PYR, GLU, ACO, GLUAC, LAC, FUM, SUC, MAL, PEP, GAHP, DHAP, GC3P, 3PGA, 2PGA, CIT, R5P, F6P, G6P, G1P, F1-6BP, S7P, SuCoA, E4P, Ru5P, X5P
Pentose Phosphate Pathway Compounds
Targeted Secondary Metabolites
Non-Mevalonate Pathway DXP, MEP, CDP-ME, MEcPP, DMAPP, HDMAPP, IPP
Phytohormones (inclu. Gibberellins) ABA, SA, JA, JA-Ile, OPDA, IAA, IAA-Asp, Methyl-IAA, IAA-Ala, IAA-Trp, t-Zeatin, c-Zeatin, t-ZRiboside, c-ZRiboside, Strigol, 5-Deoxystrigol, Orobanchol, DIMBOA, GA1, GA3, GA4, GA8, GA9, GA12, GA19, GA20, GA24, GA29, GA53
Flavonoids and Phenolic Acids Apigenin, Cyanidin, Daidzein, Genistein, Kaempferol, Luteolin, Naringenin, Phloretin, Chalconaringenin, Quercetin, Resveratrol, Caffeic Acid, Cinnamic Acid, p-Courmaric Acid, Ferulic Acid, Gallic Acid
Carotenoids Xantophyll, Zeaxanthin, Lutein, Antheraxanthin

  • Unatargeted Quantification of a wide range of small molecules
    • Primary metabolism using GC-MS and NIST/Fiehn Libraries
    • Secondary metabolism using LC-MS and Progenesis QI, NIST Library, mzCloud

Service Fees

Please contact us if you have any questions regarding the services offered and costs.

UNL Users External Academic Users Industry Users
Full Service
HPLC targeted assay $37/sample $75/sample $110/sample
LC-MS/MS targeted assay $48/sample $110/sample $170/sample
GC-MS targeted assay $42/sample $80/sample $130/sample
Protein Identification-Low Complexity $75/sample $125/sample $180/sample
Protein Identification-High Complexity $120/sample $195/sample $270/sample
Quantitative Proteomics using labeling or label-free Contact us for quote
Phosphoenrichment and Phosphoproteomics Contact us for quote
Targeted Metabolomics Contact us for quote
Non-targeted metabolomics Contact us for quote
Instrument Time UPLC $22/hour $55/hour $100/hour
Instrument Time GC-MS $25/hour $55/hour $100/hour
Instrument Time QTRAP/QE-HF $60/hour $110/hour $150/hour
Method Development/Sample Preparation $52/hour $100/hour $150/hour
Training/Data Analysis $52/hour $100/hour $150/hour
Self Service*
Instrument Time UPLC/GC-MS $15/hour $40/hour $60/hour
Instrument Time QTRAP $20/hour $55/hour $75/hour

*Self-service rates only apply to users previously trained and checked off by facility staff.

IMPORTANT Note: Scientists must contact us before submitting samples to check for compatibility of sample type and format with our analytical approaches and to confirm the experimental approach and expectations for the services.

Proteomics Platform

❖ Thermo Q-Exactive – High Field (QE-HF) coupled to Dionex U3000 nanoRSLC

  • Bottom-up proteomics for protein identification, quantification using labeling (TMT- 6 or 10 plex) or label-free approaches; PTMs characterization (phosphorylation, ubiquitination, acetylation, etc.); Intact protein characterization.
  • Targeted quantification of peptides using Parallel Reaction Monitoring (PRM) or Data Independent Acquisition (DIA).
QEHF
  • High Resolution/Accurate Mass (HR/AM) instrument
  • Resolving power up to 240,000 at 200 m/z
  • Sub ppm mass accuracy
  • Scan speed of 18 Hz at 15,000 resolution
  • Flow rate of the nanopump: 20 nL/min to 1.5 µL/min
  • Pressure to 800 bar (11,600 psi)

❖ Mascot 2.6.1 (Matrix Science Ltd) for protein identification from MS/MS spectra

❖ Proteome Discoverer 2.4 Search Engine (Thermo) for metabolic, isotope labeling and label free quantitative proteomics

❖ Scaffold 4.8.9 (Proteome Software) for protein identification, reporting and label-free quantitative proteomics

❖ PEAKS 8 (Bioinformatics Solutions Inc) for de novo sequencing and PTM characterization

Metabolomics Platform

❖ Sciex QTRAP 6500+ mass spectrometer with SelexION+ ion mobility coupled to Shimadzu Nexera II UHPLC

  • Targeted quantification of a wide range of small molecules.
QTRAP+Nexera
  • Up to 6 orders of detector dynamic range
  • Polarity switching speeds of 5 msec
  • Mass range 50-2000 m/z
  • Scan speeds of up to 20,000 Da/second
  • SelexION+ Differential Ion Mobility Technology for added selectivity
  • UHPLC with extended pressure range up to 19,000 psi for the smallest particle columns
  • Increased chromatographic reproducibility through use of micro-volume plungers
  • Autosampler has multiple rinsing lines enabling low sample carry over
  • High-speed injection

❖ Agilent 1290 Infinity II UPLC

  • Separation, profiling and quantification of a wide range of small molecules: PEG, dyes, vitamins, organic acids, carbohydrates, triglycerides, phospholipids, FAME, and more….
  • Peptide and protein fractionation for proteomics applications.
UPLC
  • Flow rate range: 0.05 to 5 mL/min
  • Pressure 1300 bar (18,800 psi)
  • Multicolumn thermostat and multisampler
  • DAD, ELSD and Fluorescence detector allowing detection of a wide range of compounds (absorbing, fluorescent, non-volatile, and semi-volatile compounds)

❖ Agilent GC-MS 5977A

  • Targeted and untargeted profiling and quantification of volatile compounds for food regulation, environmental analysis, forensic analysis, drug detection, research.
GCMS
  • Headspace sampler for analysis of volatile and semi-volatile compounds in solid, liquid and gas samples (without sample extraction)
  • NIST and Fiehn GC-MS compound libraries for identification/confirmation of unknown volatile compounds
  • Integrates with Mass Profiler Professional for qualitative and quantitative studies

Workshops & Training

Self Service Training
The facility offers training for the walk-in equipment, including the Infinity II UPLC and QTRAP 6500+. Refer to the usage and sign-up policy documents here for more information. Workshops, Seminars and Courses
Educational workshops and seminars are also organized by the facility throughout the year on a broad range of proteomics and metabolomics topics. See list below of educational workshops and seminars currently available.
  • Nebraska Center for Biotechnology - Spring Course: Intro to UNL Core Facilities and Research Methods, offered each Spring Semester
  • Women in Science
  • Nebraska Center for Biotechnology - Summer Workshop, offered each July
  • Self-Service User Digital Baging Workshops, to be offered for the first time in the summer of 2020
Past Workshops, Seminars and Courses

See list below of previous educational workshops and seminars.

Nebraska Center for Biotechnology - Summer Proteomics Workshop, July 15-16, 2019. Participants gathered to experience the full proteomics analysis pathway from sample preparation and sample running to data analysis and interpreting a final report. They were given steps for approriate preparation techniques and were shown how to best interpret reports and results. There were also hands on opportunities for those who were interested.

Anne showing participants how different protein concentrations result in different stain saturation on gels.

Anne shows participants the steps in digesting and extracing proteins from gel bands.

Participants practicing gel protein digestion and extraction techniques.

Felicia shows participants how to set up a Solid Phase Extraction (SPE) to clean protein samples.

A close up of the apparatus used for SPE.

Participants preparing to work with the SPE apparatus.

Mike showing the participants how samples are run on the Thermo Q-Exactive-HF mass spectrometer.

Mass Spectrometry-based Proteomics workshop, May 9-10, 2016. A total of 87 scientists attended the talks session in the morning of day 1, which included four experts in proteomics from various scientific fields: Michael Washburn (Stowers Institute), Hanno Steen (Harvard Medical School), Mary Lipton (PNNL) and Dmitri Nusinow (Danforth Center). Forty-two scientists, including faculty and undergraduate students registered and attended the two-day hands-on session.

Meter Nusinow

Meter Nusinow giving his talk on regulation of the circadian clock in Arabidopsis.

Students processing data

Students processing their data during the Workshop.

Students submitting samples

Attentive students submitting their samples into the mass spec instrument.

Happy students preparing samples

Students looking happy while preparing their samples.

Waters seminar : Lunch and Learn, Method Development for (U)HPLC, July 26, 2016. Participants gathered for lunch with Marguerite Arechederra and Larry Meeker, both of Waters Corporation. First Marguerite discussed appropriate column selection for various methods, from small molecules to intact proteins. Then Larry gave a case study presentation walking participants through a strategic approach to chromatography method development.

Larry Meeker explaining his approach to method development for chromatography

Larry Meeker is explaining his approach to method development for chromatography.

Waters Seminar Agenda

Personnel

Dr. Sophie Alvarez, Director
E154 Beadle Center
Phone: 402-472-4575
Email Dr. Alvarez
Publications for Dr. Alvarez

Dr. Mike Naldrett, Assistant Director
E154 Beadle Center
Phone: 402-472-4575
Email Dr. Naldrett
Publications for Dr. Naldrett

Anne Fischer, Research Technologist
E145 Beadle Center
Email Anne

Lori Loucks, Research Technician
E145 Beadle Center
Email Lori

Felicia Phares, Research Technician
E145 Beadle Center
Email Felicia

 

Publications

Listed below are the publications which our facility provided collaboration or assistance on through the services that our facility offers. For a full list of publications co-authored by individuals on our team, see the personnel page.

2019

Hankore ED, Zhang L, Chen Y, Liu K, Niu W, Guo J. Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons. ACS Synth Biol. 2019 May 17;8(5):1168-1174. PubMed PMID: 30995842. 

Graham LC, Naldrett MJ, Kohama SG, Smith C, Lamont DJ, McColl BW, Gillingwater TH, Skehel P, Urbanski HF, Wishart TM. Regional Molecular Mapping of Primate Synapses during Normal Healthy Aging. Cell Rep. 2019 Apr 23;27(4):1018-1026.e4. PubMed PMID: 31018120.

Varsani S, Grover S, Zhou S, Koch KG, Huang PC, Kolomiets MV, Williams WP, Heng-Moss T, Sarath G, Luthe DS, Jander G, Louis J. 12-Oxo-Phytodienoic Acid Acts as a Regulator of Maize Defense against Corn Leaf Aphid. Plant Physiol. 2019 Apr;179(4):1402-1415. PubMed PMID: 30643012.

Graybosch r, Bai G, Amand PS, Sarath G. Persistence of rye (Secale cereale L.) chromosone arm 1RS in wheat (Triticum aestivum L.) breeding programs of the Great Plains of North America. Gen Res Crop Evol. 2019 Apr 1;66 (4):941-950. Link to Article.

Kim WS, Krishnan HB. Impact of co-expression of maize 11 and 18 kDa δ-zeins and 27 kDa γ-zein in transgenic soybeans on protein body structure and sulfur amino acid content. Plant Sci. 2019 Mar;280:340-347. PubMed PMID: 30824013.

Shihabudeen Haider Ali MS, Cheng X, Moran M, Haemmig S, Naldrett MJ, Alvarez S, Feinberg MW, Sun X. LncRNA Meg3 protects endothelial function by regulating the DNA damage response. Nucleic Acids Res. 2019 Feb 20;47(3):1505-1522. PubMed PMID: 30476192.

Zapf RL, Wiemels RE, Keogh RA, Holzschu DL, Howell KM, Trzeciak E, Caillet AR, King KA, Selhorst SA, Naldrett MJ, Bose JL, Carroll RK. The Small RNA Teg41 Regulates Expression of the Alpha Phenol-Soluble Modulins and Is Required for Virulence in Staphylococcus aureus. mBio. 2019 Feb 5;10(1). PubMed PMID: 30723124.

Shah SB, Parmiter D, Constantine C, Elizalde P, Naldrett M, Karpova TS, Choy JS. Glucose Signaling Is Connected to Chromosome Segregation Through Protein Kinase A Phosphorylation of the Dam1 Kinetochore Subunit in SaccharomycescerevisiaeGenetics. 2019 Feb;211(2):531-547. PubMed PMID: 30546002.

Pan M, Li M, Li L, Song Y, Hou L, Zhao J, Shen B. Identification of Novel Dense-Granule Proteins in Toxoplasma gondii by Two Proximity-Based Biotinylation Approaches. J Proteome Res. 2019 Jan 4;18(1):319-330. PubMed PMID: 30362762.

Palmer NA, Basu S, Heng-Moss T, Bradshaw JD, Sarath G, Louis J. Fall armyworm (Spodoptera frugiperda Smith) feeding elicits differential defense responses in upland and lowland switchgrass. PLoS One. 2019;14(6):e0218352. PubMed PMID: 31194847.

2018

Wang X, Edwards RL, Ball H, Johnson C, Haymond A, Girma M, Manikkam M, Brothers RC, McKay KT, Arnett SD, Osbourn DM, Alvarez S, Boshoff HI, Meyers MJ, Couch RD, Odom John AR, Dowd CS. MEPicides: α,β-Unsaturated Fosmidomycin Analogues as DXR Inhibitors against Malaria. J Med Chem. 2018 Oct 11;61(19):8847-8858. PubMed PMID: 30192536.

Chapman KM, Marchi-Werle L, Hunt TE, Heng-Moss TM, Louis J. Abscisic and Jasmonic Acids Contribute to Soybean Tolerance to the Soybean Aphid (Aphis glycines Matsumura). Sci Rep. 2018 Oct 11;8(1):15148. PubMed PMID: 30310120.

Beltrán J, Wamboldt Y, Sanchez R, LaBrant EW, Kundariya H, Virdi KS, Elowsky C, Mackenzie SA. Specialized Plastids Trigger Tissue-Specific Signaling for Systemic Stress Response in Plants. Plant Physiol. 2018 Oct;178(2):672-683. PubMed PMID: 30135097.

Garcia Tavares R, Lakshmanan P, Peiter E, O'Connell A, Caldana C, Vicentini R, Soares JS, Menossi M. ScGAI is a key regulator of culm development in sugarcane. J Exp Bot. 2018 Jul 18;69(16):3823-3837. PubMed PMID: 29767776.

Shang X, Chen Y, Wang N, Niu W, Guo J. Oxidation-induced generation of a mild electrophile for proximity-enhanced protein-protein crosslinking. Chem Commun (Camb). 2018 Apr 19;54(33):4172-4175. PubMed PMID: 29629441.

Long S, Brown KM, Sibley LD. CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii. Bio Protoc. 2018 Mar 20;8(6). PubMed PMID: 29644258.

Kesinger E, Liu J, Jensen A, Chia CP, Demers A, Moriyama H. Influenza D virus M2 protein exhibits ion channel activity in Xenopus laevis oocytes. PLoS One. 2018;13(6):e0199227. PubMed PMID: 29927982.

2017

Long S, Anthony B, Drewry LL, Sibley LD. A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii. Nat Commun. 2017 Dec 21;8(1):2236. PubMed PMID: 29269729.

Liu K, Enns B, Evans B, Wang N, Shang X, Sittiwong W, Dussault PH, Guo J. A genetically encoded cyclobutene probe for labelling of live cells. Chem Commun (Camb). 2017 Sep 21;53(76):10604-10607. PubMed PMID: 28902227.

Ross KC, Andrews AJ, Marion CD, Yen TJ, Bhattacharjee V. Identification of the Serine Biosynthesis Pathway as a Critical Component of BRAF Inhibitor Resistance of Melanoma, Pancreatic, and Non-Small Cell Lung Cancer Cells. Mol Cancer Ther. 2017 Aug;16(8):1596-1609. PubMed PMID: 28500236.

Long S, Brown KM, Drewry LL, Anthony B, Phan IQH, Sibley LD. Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii. PLoS Pathog. 2017 May;13(5):e1006379. PubMed PMID: 28475612.

Belayneh HD, Wehling RL, Reddy AK, Cahoon EB, Ciftci ON. Ethanol-modified supercritical carbon dioxide extraction of the bioactive lipid components of Camelina sativa seed. Seed. J Am Oil Chem Soc. 2017 Apr;94:855-865. Link to Article.

Belayneh HD, Wehling RL, Cahoon EB, Ciftci ON. Lipid composition and emulsifying properties of Camelina sativa seed lecithin. Food Chem. 2017 Aug;242:139-146. Link to Article.

Khasin M. Phytohormone signaling in Chlorella sorokiniana: perspectives on the evolution of plant cell-to-cell signaling Dissertations and Theses in Biological Sciences. 2017 Apr;95. Link to Article.

Gulati P, Li A, Holding D, Santra D, Zhang Y, Rose DJ. Heating Reduces Proso Millet Protein Digestibility via Formation of Hydrophobic Aggregates. J Agric Food Chem. 2017 Mar 8;65(9):1952-1959. PubMed PMID: 28198183.

Belayneh HD, Wehling RL, Cahoon EB, Ciftci ON. Effect of Extraction Method on the Oxidative Stability of Camelina Seed Oil Studied by Differential Scanning Calorimetry. J Food Sci. 2017 Mar;82(3):632-637. PubMed PMID: 28182838.

Shang X, Song X, Faller C, Lai R, Li H, Cerny R, Niu W, Guo J. Fluorogenic protein labeling using a genetically encoded unstrained alkene. Chem Sci. 2017 Feb 1;8(2):1141-1145. PubMed PMID: 28451254.

Deniz Ciftci, PhD Thesis: Subcritical Water-Assisted Fractionation of Lupin Hull for Production of Cellulose Nanofiber Hydrogels and Aerogels. 2017. Link to Article.



Advisory Committee

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