Sample Preparation

The method of sample preparation largely depends on the aim of the research, yet the method is key to the success of experiments. While it is essential to reduce the complexity of a mixture, this process can be the most difficult part of proteomics research. There are many sample preparation options and methods available. Here are some precautions/recommendations for sample preparation:

HPLC protein and peptide purification:

• Samples submitted should be free of non-volatile buffer components if possible.

• For an analytical HPLC separation, up to 500 µg protein can be separated. An estimate of the quantity and quality of starting material is needed.

• We recommend samples dissolved in 0.1% TFA. If not soluble, add less than 15% acetonitrile.

• For special needs, columns and separation methods should be provided by users.

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.

Mass spectrometry:

• The Voyager DE STR and QSTAR are of superior resolution and sensitivity. An amount as little as 150 fmol may be enough for a protein identification. However, we would recommend 1-5 pmols for high quality data (10 ng for a 10 kDa protein is 1 pmol).

• 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 (small amount of phosphate, Tris and NaCl may be tolerated in MALDI mode).

• Avoid glycerol, DMSO, SDS, urea and guanidine, especially for MALDI analysis. If detergents must be used, octyl glucoside (0.1%) is the best choice.

We provide the following services for removing contaminants and interfering substances: Microcon centrifugal filteration (3 kDa cut-off), Mini-dialysis, TCA acetone precipitation, C18 or C4 micro-reversed-phase chromatography (Zip-tipping), and Amersham 2D clean-up. When submitting samples, please use a sample container of appropriate size. Two µL of sample is more easily recovered from 0.5mL Eppendorf tubes than from 1.5mL . Prerinsing the tube with methanol or acetonitrile lowers the chemical background for low concentration samples or complex mixtures. Please include controls (positive and/or negative) if possible (e.g. submit trypsinized gel plug without protein when submitting in-gel digested samples).

return to top

Gel Electrophoresis

Reservations for 2D gel usage (accessible by Danforth Center employees only)

Polyacrylamide gel electrophoresis (PAGE) proves to be a very useful technique for protein separations. When run under denaturing conditions in the presence of sodium dodecyl sulfate (SDS; lauryl sulfate), great reproducibility has been achieved. One dimensional (1D) SDS-PAGE is used to determine the relative abundance, approximate molecular weights, and relative purity of major proteins in a sample. For complex samples, 1D SDS-PAGE suffers from low resolution and limited sample capacity. To solve these problems, researchers have invented different versions of 2 and 3 dimensional PAGE techniques to serve different research purposes.

1. SDS-PAGE: proteins are separated according to molecular mass.  This is good for simple samples and hydrophobic proteins.

2.  Isoelectric focusing (IEF) + SDS-PAGE: proteins are first separated by isoelectric point (pI), and then by molecular weight (MW) in the second dimension. 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. With the commercialization of immobilized pH gradient (IPG) strips of various pH gradients and precast gels of different ranges, 2D PAGE has become highly reproducibile and very powerful, allowing the ‘zoom out’ and ‘zoom in’ of proteins in the sample. 2D PAGE starts to show its limitation when analyzing very large (> 100 kDa) or small proteins (< 5 kDa), low abundant proteins, proteins with extreme pIs, and certain membrane proteins.

3. Blue native PAGE (BN) + SDS-PAGE: proteins are first separated in native forms, and then the gel lane is excised and put on top of SDS-PAGE runs in the second dimension. This procedure is very useful for membrane and soluble protein complexes.

4. BN + IEF + SDS-PAGE: This 3D combination provides high resolution separation of protein complexes.

We provide 1D and 2D gel services. When the needs arises, we will consider 3D gels.  For quantitative analysis, 5 replicate gels (or at least 3 replicates) should be run per sample. We encourage and willingly provide support to internal users to run and process gels in the facility.

return to top

Imaging

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.

1. Colloidal Coomassie Blue (Bio-Safe Coomassie): it stains the broadest spectum of proteins. It has about 2 orders of magnitude and sensitivity down to 10 ng.

2. 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. It also allows for detection of glycoproteins, lipoproteins, low MW proteins and metalloproteins that do not stain well by other stains. The excitation peaks of the gel stain are at 280 nm and 450 nm, and the emission maxima is near 610 nm.

3. 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.

4. Negative Staining: copper-, zinc- or imidazole-zinc-staining can be used for eluting whole proteins from SDS-PAGE and subsequent mass spectrometry analysis (Anal. Biochem. 1997, 247: 257-267).

Images from gels stained with Coomassie and Silver can be acquired with a flatbed scanner (Epson Expression 1640XL). For Sypro stained gels, a Typhoon 9410 imager is used. The digitized gel images are saved as TIFF files.

After image acquisition of replicate gels of different samples, the images need to be analyzed using computer-assisted gel image analysis software which does spot detection, matching, spot normalization and quantitation, pI and MW determination, gel average and comparison, statistical analysis, annotation and documentation. Gel image analysis usually is a time-consuming process.

If gels are not going to be analyzed further, they can be archived and stored for long periods of time at 4° C.

return to top

Protein Spot Excision

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.

return to top

Protein Digestion

Proteins are usually digested into peptides for proteomics analysis. It is useful to reduce/alkylate your protein samples prior to proteolytic digestion because attempts to digest mature proteins composed of disulfide-bonded structures are often unsuccessful. Of the many enzymes with different specificities used for digestion of proteins, trypsin is the most common. Other enzymes or chemicals such as cyanogen bromide (CNBr) can be chosen depending on proteins and purposes. While manual in gel or in solution digestion tends to give better yield, automated digestion performed by MultiProbe II digester in a temperature-controlled enclosed environment gives higher reproducibility and provides less contamination. The built-in program allows for overnight tryptic digestion of up to 2 x 96 samples, greatly reducing the labor component of the digestion procedure. Please consider automatic digestion if you have more than 16 samples.

return to top

MALDI-TOF Analysis

Reservations for Maldi-TOF usage (accessible by Danforth Center employees only)

MALDI-TOF stands for matrix assisted laser desorption/ionization-time of flight. MALDI is a type of soft ionization. The analyte is first co-crystallized with a UV-absorbing matrix (which acts as a proton donor or acceptor), then subjected to pulse laser radiation. This causes the vaporization of the analyte/matrix crystals and produces ions which are directed into a flight tube. The mass of an ion is measured by the time it takes to arrive at the detector (smaller ions are faster, larger ions are slower). The time is then converted to mass to charge ratio.

The Voyager DE STR Biospectrometry Workstation in our facility is an advanced MALDI-TOF instrument featuring a long flight path (3 meter), high quality ion optics, and upgraded electronics. Its reflectron provides very high resolution and mass accuracy. It has the capability to analyze a mixture of compounds without separation. Voyager software with Data Explorer integrates instrument operation and data processing functions.  Here are some instrument specifications:

Voyager can be used in many applications:

• Molecular weight determination

• Peptide fingerprinting for protein identification

• N-terminal protein and oligonucleotide sequencing using In-Source Decay (ISD)

• C-terminal protein and peptide sequencing (Post-Source Decay (PSD))

• Post translational modifications: glycolysation, phosphorylation, sulfation

• Carbohydrate analysis

• Small molecule analysis

Although MALDI is good for the analysis of complex mixtures, samples for MALDI should be as pure as possible (no detergent and phosphate), and have very low concentrations of salts, glycerol, urea, guanidine, tris, etc.  With the use of both the SymBiot and the MultiProbeII robot to spot MALDI plates, the Voyager supports multiple workflow schemes, including conventional chromatography and ICAT analysis. If there is a large batch of samples, the Voyager is available in a self-service format after training by facility staff. Contact us for training opportunities.

return to top

Q-TOF Analysis

We have the hybrid Q-TOF (quadrupole time-of-flight) QSTAR Pulsar XL MS/MS system from the Applied Biosystems.  The QSTAR design permits the use of a variety of ionization sources, especially electrospray. Electropray is a type of soft ionization. When a sample is introduced, a positive DC voltage (2.5-5 kv) is applied to the spray needle (sharply pointed hollow metal tube), creating charged droplets of solvent. As the solvent evaporates, the charge density increases, which creates repulsion and droplet dissociation. Further evaporation creates a charge transfer from the solvent to the analyte, generating charged ions, which then move into the mass analyzer through differential pumping.  A major feature of electrospray is that it creates multiple charged ions.

The QSTAR® XL Hybrid LC/MS/MS System is a high-performance mass spectrometer which generates superior quality MS and MS/MS data for protein identification/characterization and drug metabolites. It has the following features:

• Enhanced ion optics for highest sensitivity and reliability

• Excellent mass accuracy and stability yield unequivocal molecular weight and high-quality structural information

• Unique LINAC™ Pulsar collision cell technology enables the most sensitive product ion and precursor ion scan capabilities for metabolite, protein and peptide, and post-translational modification determination

• Maximum flexibility with a comprehensive selection of interchangeable, application-specific ion sources including electrospray (ESI), i.e. the TurboIonSpray and NanoSpray (for extremely low flow rates for small quantities of samples), and orthogonal MALDI with a higher tolerance for impurities.  This is an advantage for working with a variety of sample preparations.

NanoSpray ionization on the QSTAR is very sensitive, as is MALDI, usually only requiring 1 to 10 pmol of samples. The mass limit for the quadrupole is 3 kDa for singly charged ions, and extends to 3 kDa times the number of charges the ion carries. For those requesting reverse phase separation prior to mass spectrometer analysis, we have an LC Packings UltiMate™ nanoliter flow HPLC on the front end of the QSTAR for medium-throughput LC/MS/MS analysis. Here are some technical specifications:

 The QSTAR allows for the following sensitive analyses:

• NanoESI accurate protein molecular weight determination

• Accurate oMALDI mapping of peptides, oligosaccharides and glycoconjugates

• Biopolymer sequence analysis (m/z <3,000)

• High-throughput peptide sequencing

• Sensitive precursor and product ion scanning (allowing identification of the type and location of post-translational modifications)

• Powerful information dependent acquisition (IDA) experiments

• Phosphopeptide analysis

• Glycopeptide analysis

• ESI softest ionization allows for observing protein-ligand, protein-protein, protein-nucleic acid and other native noncovalent interactions.

• Small molecules and drug discovery

Samples to be directly analyzed using the QSTAR in ESI mode should be pure and free of salt. They can be submitted as liquid, e.g. in 5% acetonitrile, or 0.1% TFA, or as dried material. Separation methods other than those discussed for protein/peptide samples are to be provided by users. Due to the sophisticated nature of operating the instrument, we only provide full service for QSTAR samples.

return to top

Other Services

Biacore 2000 (Biacore Inc., Surface Plasmon Resonance Technology), Gold HPLC (Beckman Coulter,with autosampler, diode array and fluorescence detector) and BioCad 700E Perfusion Chromatography Workstation (Applied Biosystems, with a pH meter and conductivity cell in addition to a UV/VIS detector) are currently available for customer self-service. Customers need to obtain necessary training prior to using the instruments. Please contact us for more information.

return to top