Proteomics & Mass Spectrometry

The Danforth Center’s Proteomics & Mass Spectrometry Facility develops new tools and provides high quality sample analysis in proteomics, mass spectrometry and related analytical fields.

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Services

Please contact us to discuss your project goals. We will try to accommodate you if necessary instrumentation and resources are available for services not described above.

Training for self-service users is provided for select offerings for a fee.

Sample Preparation    TOP

We accept samples prepared from customers but they have to meet some requirements. Please read recommendations below:

For HPLC protein and peptide purification:

• Samples submitted should be free of non-volatile buffer components if possible.
• For special needs, columns and separation methods should be provided by users.

For 2-D gel protein separation:

• For analytical purposes, as little as 50 µg protein is needed. For preparative purposes, more than 1 mg protein can be loaded. The amount of sample depends on the complexity and the purpose of research.
• Salts, residue buffer components, and other charged small molecules should be removed. The tolerance for salts is 10 mM.
• Use zwitterionic or non-ionic detergents to increase protein solubility.
• Samples rich in nucleic acids should be treated with protease-free DNase/Rnase.
• Polysaccharides, lipids, and phenolic compounds should all be removed if possible.

For mass spectrometry:

• Use volatile, salt-free solvents such as methanol and acetonitrile.
• Salts normally form adduct peaks which suppress the molecular ion signal. Exchange sodium and potassium for ammonium when possible. Avoid phosphate buffers, use minimum concentrations of ammonium bicarbonate or ammonium acetate to control pH
• Avoid glycerol, DMSO, SDS, urea and guanidine. If detergents must be used, octyl glucoside (0.1%) is the best choice.

We also provide the following services:

• Protein extraction using phenol followed by methanolic ammonium acetate precipitation.
• For better homogenization of samples, the samples can be ground using the TissueLyser II
• For removing contaminants and interfering substances: TCA acetone precipitation, C18 or C4 micro-reversed-phase chromatography (Zip-tipping) Centrifugal filter and Perfect Focus 2D clean-up.

Gel Electrophoresis and Gel Staining    TOP

We provide 1D and 2D gel services. We encourage and willingly provide support to internal users to run and process gels in the facility.

• 1D-SDS-PAGE proteins are separated according to molecular mass. This is good for simple samples and hydrophobic proteins.
• 2D-Gels Here, the initial protein separation is done through isoelectric focusing using the BioRad Protean IEF Cell or IPGphor from Amersham Biosciences. Proteins are then separated by molecular mass using Dodeca Cell from BioRad or Ettan Daltsix system from Amersham Biosciences. This is a traditional 2D PAGE, which can resolve a large numbers of proteins (up to 2000) on a single gel. When stained with dyes of high sensitivity and dynamic range, protein expression levels can be quantified, thus enabling global protein expression analysis.

Imaging and Image Analysis    TOP

Proteins in gels must be stained or labeled in order to visualize the proteins. The facility uses several stains, the choice of which depends on the downstream mass spectrometric analysis.

• Colloidal Coomassie Blue (Bio-Safe Coomassie): it stains the broadest spectrum of proteins. It has about 2 orders of magnitude and sensitivity down to 10 ng.
• SYPRO Ruby fluorescent staining: it gives little background staining and is very sensitive (1 ng). The stain is linear over 3 orders of magnitude which is very useful for quantitative analysis. The excitation peaks of the gel stain are at 280 nm and 450 nm, and the emission maxima is near 610 nm.
• Silver Staining: it is very sensitive (1 ng), but the linearity is low (1 order magnitude). Be aware that traditional silver staining involving oxidization of proteins is not compatible with mass spectroscopic analysis since the oxidative step changes protein mass. As a result, mass spectrometric compatible silver staining procedures, such as SilverQuest from Invitrogen or Silver Stain Plus from BioRad, should be used.
• ProQ Emerald glycoprotein stain reacts with periodate-oxidized carbohydrate groups, creating a bright green-fluorescent signal on glycoproteins. It is possible to detect as little as 0.5 ng of glycoprotein per band, depending upon the nature and the degree of glycosylation. The stain can be visualized with 300 nm UV illumination. After imaging, the proteins can be counter stained with SYPRO ruby to detect all the proteins.
• ProQ Diamond stain allows direct, in gel detection of phosphate groups attached to tyrosine, serine, or threonine residues. The stain allows detection of as little as 1-16 ng of phosphoprotein per band, depending on the phosphorylation state of the protein. ProQ Diamond has excitation/emission maxima of 555/580 nm. After imaging, the proteins can be counter stained with SYPRO ruby to detect all the proteins.
  
  Images from gels stained with Coomassie and Silver can be acquired with a flatbed scanner (Epson Expression 1640XL). For Sypro and ProQ Diamond stained gels, a Typhoon 9410 imager is used. The ProQ Emerald stained gels can be imaged using a UV transilluminator.
  
  After image acquisition of replicate gels of different samples, the images need to be analyzed using computer-assisted 2D gel image analysis software (Progenesis SameSpot) which does spot detection, alignment, matching, spot normalization and quantitation, pI and MW determination, gel average and comparison, statistical analysis, annotation and documentation.

Protein Spot Excision and Digestion     TOP

Protein bands or spots in PAGE gels need to be excised before the proteins can be identified. When cutting them manually, please excise as close to the band or spot as possible. Large gel bands need to be cut further into 1 mm squares. Prevent keratin and dust contamination by wearing gloves, lab coats, masks, etc. Avoid sample cross-contamination. For low abundant proteins, you may need to combine spots. Our facility is equipped with a high throughput and accurate spot picker (GelPix) that can pick spots from Silver or Coomassie stained gels as well as from Sypro stained gels..

Proteins are usually digested into peptides for proteomics analysis. While manual in gel or in solution digestion tends to give better yield, automated digestion performed by MultiProbe II Instrumentation digester in a temperature-controlled enclosed environment gives higher reproducibility and provides less contamination.

Protein Identification using LC-MS/MS and Database Searching     TOP

Typically, protein digests are submitted to LC-MS/MS for protein identification. The facility is equipped of 3 mass spectrometers that can be used to acquire peptide information:

• QSTAR XL Q-TOF mass spectrometer (Applied Biosystems) online with nano-flow LC (LC Packings or Eksigent NanoLC-2D
• 6520 QTOF  mass spectrometer (Agilent) online with nanoLC-ChipCube (Agilent).
• LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific) online with either the nanomate Triversa (Advion) or the nano-flow LC Eksigent Ultra Instrumentation
  
  The acquired data from any of the platform can be submitted to database searching and protein identification using MASCOT v2.2 database searching engine. We also have an additional bioinformatics tools for data conversion (MASCOT Distiller) and for protein reporting (Scaffold 2.0, Proteome Software).

Quantitative Proteomics (Labeling Gel-based and LC-based) using DIGE and iTRAQ    TOP

We offer labeling quantitative proteomics using DIGE and iTRAQ:

• Difference Gel Electrophoresis (DIGE) is a modification of 2-D PAGE. We use CyDye TM DIGE Fluor dyes (Cy2, Cy3, and Cy5). Two separate protein samples are labeled with different fluorescent dyes Cy3 and Cy5 prior to separation. Cy2 is used to label the internal standard which contains half of the proteins from each sample. The three samples are then combined to run into a single gel, enabling accurate analysis of differences in protein abundance between samples. Downstream of this, technologies available in the facility include: Cy-dye labeling, isoelectric focusing using Immobilized pH Gradient strips, 2nd dimension SDS gel electrophoresis, fluorescence scanning, image analysis with SameSpot software package, and protein identification of desired spots.
• iTRAQ (isobaric tag for relative and absolute quantitation) is a non-gel based technique used to identify and quantify proteins from different sources in one single experiment. It uses isotope coded covalent tags. The method is based on the covalent labeling of the N-terminus and side chain amines of peptides from protein digestions with tags of varying mass. The facility is set up to run the 4-plex iTRAQ which can be used to label all peptides from 4 different samples/treatments. After labeling the samples are pooled and fractionated by HPLC (System Gold from Beckman/Coulter) using SCX. Each fraction is then analyzed by LC-MS/MS. A database search is then performed using ProteinPilot (Applied Biosystems). The fragmentation of the attached tag generates a low molecular mass reporter ion that can be used to relatively quantify the peptides and the proteins from which they originated.

Targeted Analysis of Small Molecules     TOP

The facility is equipped with several instruments for targeted analysis of small molecules including an Agilent 5975C Series GC/MSD. This instrument is ideal for the analysis of volatile and semi-volatile organic compounds in mixtures. Additionally, we have a 4000 QTRAP LC/MS/MS system (Applied Biosystems), which is a hybrid triple quadrupole/linear ion trap mass spectrometer ideal for drug discovery and development, and metabolite identification. A Shimadzu HPLC using an auto sampler from Leap Technologies is coupled to the QTRAP instrument for separation of complex samples and metabolite quantification. The following targeted methods are offered as full service:

Acidic Plant Hormone Analysis    TOP

We are using the 4000 QTRAP LC-MS/MS system for targeted analysis and quantitation of the following acidic plant hormones: Jasmonic acid (JA), Salicyclic acid (SA), Abscisic acid (ABA), Indole-3-acetic acid (IAA), JA-Ile (jasmonoyl-isoleucine), OPDA (12-oxo-phytodienoic acid), IAA-Asp (Indole-3-acetyl-aspartic acid). For more information, please see PlantHormone.

Amino Acid Analysis     TOP

We are using the AccQ•Tag system from Waters for derivitization followed by separation on a Waters Acquity UPLC system. It is ~9.5 min separation and we do either free amino acids (client extracted) or HCl hydrolysis (usually from plant tissue). We recommend samples be run in triplicate for appropriate statistical analysis. The chemistry is based on Waters' widely-used and understood AccQ•Tag derivatizaton method. Primary and secondary amino acids react with a single reagent, AccQ•Fluor™, in a high-throughput batch process resulting in exceptionally stable derivatives. High resolution separations are achieved using pre-qualified AccQ•Tag Ultra UPLC columns and mobile phases. Derivatized amino acids are quantified to sub-picomole levels with single wavelength UV detection. For more information, please see AAA.