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FAQ's
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General:
What is the best method for protein identification?
In the first instance we strongly recommend mass spectrometry (MS/MS) for protein identification, especially if there is a chance that the protein is blocked at the N-terminus. This is the state-of-the-art technique for obtaining internal sequence information, and with an appropriate strategy, can be used to sequence entire proteins. This technique can readily provide sufficient data to show homology with other proteins, or for novel proteins to obtain internal amino acid sequence information to enable cloning of the gene. The alternative approach of traditional N-terminal Edman sequencing is less sensitive and slightly more expensive. It has the benefit of providing the definitive sequence of the N-terminus.
Frequently Asked Questions about Mass Spectrometry:
How sensitive is analysis by MS/MS (MALDI-TOF/TOF or LC/MS/MS)?
The technique is more sensitive than N-terminal Edman sequencing. This method can also use the protein directly from an SDS-PAGE gel. The band or spot can be stained with Coomassie and excised into a small sample tube and dried. The protein is then relatively stable and can be sent by post/courier. Most bands visible by Coomassie staining will produce an identification. Protein identification is also possible from silver or Sypro stained proteins (see below).
What amount of protein is required?
For a pure protein: 100ng-1µg.
How can I avoid keratin contamination?
Best results are always obtained from clean samples. Contamination occurs primarily from keratin proteins from hair and skin. To avoid keratin contamination the use of gloves when handling samples is recommended.
Is pooling required for silver or Sypro stained gels?
Yes. Providing as much protein as possible increases the chances of a successful identification. For silver we recommend pooling two spots, and for Sypro ruby or Cy dyes a minimum of 3. For low abundance samples of this type our high sensitivity nano-LC MS/MS instrument will be used.
What is the treatment required for Sypro ruby stained, silver stained or Coomassie stained gels respectively?
All proteins will be destained by us prior to analysis. No pre-treatment is required by yourself. However, the type of stain used is important:
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For Coomassie there are no extra requirements. -
For silver stain, a mass spectrometry compatible procedure must be used. Any gels stained with a traditional silver stain method that uses gluteraldehyde cannot be analysed by mass spectrometry. -
Several suppliers offer silver stain kits that are mass spec compatible (e.g. Amersham International: Plus-One Staining Kit). -
Sypro ruby is compatible with mass spectrometry analysis, however, pooling of bands is required.
How should the sample be prepared?
The protein can be sent as a band or spot excised from an SDS-gel (either stained with Coomassie or MS compatible silver stain). The gel piece should be air dried in a microcentrifuge tube. Care must be taken to ensure no contamination of the bands or spots with human proteins (skin) or dust. We will trypsin digest the proteins.
PI will trypsin digest the proteins.
Note: Proteins on PVDF membrane cannot be analysed by mass spectrometry.
I have an old sample, can it still be analysed?
PLEASE NOTE that silver stained gels older than 1-2 months may not yield satisfactory results. Coomssie stained spots and bands can be analysed even when several months old.
Can samples be analysed in liquid form?
Yes if the sample is pure. The sample must be freeze dried or air dried in a micro-centrifuge tube prior to shipping.
[Note. Complex liquid samples containing several proteins can also be analysed in some circumstances.
Please consult us before submitting samples in this case].
There is no price difference for MS analysis of proteins from gel pieces or liquid samples.
SERVICES
Protein Identification by Mass Spectrometry:
Protein identification by mass spectrometry (Mass spectrometry of gels slices by MS/MS/TOF)
For protein sequencing by tandem time-of-flight MS (MALDI or LC MS/MS) the protein would be digested into peptides and a spectra obtained for the major peptide ions.
The sample spectra is compared to databases to look for identical sequences. This can be adequate to identify a known protein, or closely related proteins.
[For samples which cannot be identified by automatic database analysis please refer to Service 002 - De novo peptide sequencing form 002
The sample spectra is interpreted to obtain de novo protein sequence for each peptide ion. This is the equivalent of N-terminal Edman sequencing of internal peptides and, if successful gives the highest quality data. The sequences can range in length from 6-20+ amino acids and these can be used to design a probe (the identification of several peptides may be required to produce a sequence that best suits design of an oligo probe)].
Sample prep: See entire section "Frequently asked Questions on Mass Spectrometry" above
Use of protein identification by MS/MS
The sample must be from a known genome, or for which a closely-related genome that has been sequenced. Please specify the source genome.
We need to know:
1. Is this pure protein?
2. Is protein present in currently available databases?
Cost:
For One sample and/or Bulk analysis (>50) see Pricing
Please note:
No result: $ 50 per sample
All prices are in US Dollars.
Quarantine inspection fees may be incurred. Searches are random and cannot be avoided. Where charges are incurred these will be passed on to the client. Where fees are incurred due to incorrect labeling this will be charged to the client (maximum $100 per package).
Results:
Reports are supplied electronically (only) containing web-links to view individual results upon completion, for example see
Results are held on a secure server where clients have password access to their data area.
Raw data files (compatible with Mascot) are supplied to enable user specific search parameters. Hard copies of reports and data, if required will only be supplied upon payment of invoice (hard copy surcharge US$20), however it will only contain the http address specific for each sample.
Delivery time
15 working days from receipt of sample
Peptide mass fingerprinting by MALDI-TOF mass spectrometry:
This is a more basic technique for protein identification. It has been superseded by our protein identification service 001 (PMF+MS/MS)
Please download form 001
How should samples be prepared from gels?
The protein should be electrophoretically transferred to a good quality (protein sequencing grade) PVDF membrane. Such membranes are available from Bio-Rad, Applied Biosystems, GE and others. The membrane should be stained with Coomassie Blue, and the target band must then be visible by eye. The entire membrane should be sent intact along with a photocopy indicating the band to be sequenced. PI will excise the band.
Please ensure the blots are free of any particulate material.
PLEASE NOTE: this service cannot be used for proteins in SDS-gels.
Can samples be analysed from liquid form?
Yes, however the sample must be freeze dried in a micro-centrifuge tube prior to shipping.
Sample should be free of interfering buffers and salts. Buffers containing primary amines, such as TRIS and HEPES, should be removed, although low concentrations of SDS (up to 0.3%) and PBS can be tolerated.
[Note. Complex samples containing more than one protein cannot be analysed by this process].
What amount of protein is required?
For a pure protein the sensitivity limit is approximately 1 picomole, however we recommend 10pmol to ensure a satisfactory result.
What if the protein is blocked at the N-terminus?
No sequence information will be obtained and the “no result” fee applied. The only alternative is to attempt internal sequencing in which case mass spectrometry based analysis is used.
Can Cysteine be detected?
Cysteine without special modification can not be detected by N-terminal sequencing and will give a blank result. The sample has to be modified for detection of cysteine.
Note: Sample should be freeze-dried or in solution.
Please indicate if you require Cys analysis.
Two specific points to note include:
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Protein band must be visible by Coomassie Blue staining. -
The membrane should be sent intact, along with a photocopy indicating the band to be sequenced.
General Questions:
The sample is on PVDF membrane, is it ok?
No, the protein must be lyophilised.
The sample is in liquid solution - is it OK?
No, please freeze dry prior to shipping to ensure maximum stability.
What quantity of proteins is required?
Our HPLC detection limit is 500 pmole per injection. With sample handling limitations this means we require nmoles of the target peptide. For proteins we require 100x this amount.
What to avoid when preparing a sample for amino acid analysis?
Purity of the sample is critical. This essentially means that 100% protein purity is required to obtain an accurate result. A 10% protein contamination can corrupt your final result.
Contamination with proteins, amino acids, buffers and salts will interfere with the analysis. Buffers containing primary and secondary amines, such as Glycine, Tris, HEPES and glycerol must be removed. These materials may be removed by ethanol-precipitation, trichloroacetic acid precipitation, reverse-phase extraction, gel filtration, ion-exchange separation, or dialysis.
What are the main problems encountered?
Cysteine and tryptophan cannot be analysed by conventional acid hydrolysis. They are destroyed in the hydrolysis process and require special hydrolysis methods.
Please indicate if you require cysteine and tryptophan analysis.
Asparagine and glutamine are 100% converted to aspartic and glutamic acids during acid hydrolysis of proteins and cannot be measured independently in a protein sample.
We require details on:
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Volume before drying -
Solvent/buffer used -
Known or estimated concentration of sample
Delivery
20 working days
We provide a comprehensive service from crude protein extract to image analysis of client gels. If a sample is provided it should be in the form of a crude protein extract pellet.
How many gels are required?
A minimum of 3 replicate gels must be run for each sample, and normally 4 or 5 are required. Replicate images are then combined to form a proteome master gel using specialist software.
What amount of sample is required?
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1-2mg total protein (assuming a complex protein mixture) is required for a large format (20cm) gel stained with Coomassie. -
0.3-0.5 mg total protein (assuming a complex protein mixture) is required for a small format (12cm) gel stained with Coomassie.
Other stains are possible which require smaller amounts of protein, but their use depends on the desired outcome.
A guide to prices is on this website, however, please note the final price will depend on the number of gels required. Frequently 2-5 test gels are needed to optimise the method, and then we recommend quadruplicate gels for comparison of samples.
Analysis by 2D gel electrophoresis is complex and we insist on full details in advance to avoid client disappointment. Please provide details of the desired experimental outcome if further information is required.
All work is subject to the company's standard terms and conditions
Delivery
20+ working days (project specific)
Protein quantitation by iTRAQ:
iTRAQ enables relative and absolute quantitation of protein and peptides by labeling samples with isotope encoded reporter ions.
The protein sample is acetone precipitated, digested and labeled. Four different samples can be labeled with iTRAQ reagents. Labeled peptides are first separated by strong cation exchange chromatography. Each cation exchange fraction is further analysed by reverse phase LC-MALDI. Results are compared with special software.
What amount of protein is required?
The optimal amount for an iTRAQ experiment is 50µg tagged protein. Losses during sample cleanup can be considerable and are sample specific. We therefore recommend that 200-500µg of sample is provided; greater amounts will improve sensitivity for low abundant proteins.
What type of samples can be analysed?
Cell lysates, secreted protein samples, but not radioactive samples.
#Note: secreted samples must be in serum/plasma free media.
What is the maximum volume of sample?
The sample volume must be less than 250µl.
Can I use protease inhibitors?
Yes, Protease inhibitors will minimise protein degradation.
Is it important that your proteome that you are looking at is in currently available databases?
Yes, as iTRAQ analysis will not work on samples from an unknown genome.
Delivery
20 working days
Multiple Reaction Monitoring (MRM):
What is MRM?
Multiple reaction monitoring is a highly specific and sensitive mass spectrometry technique that can selectively quantify compounds within complex mixtures. This technique uses a triple quadrupole MS that firstly targets the ion corresponding to the compound of interest with subsequent fragmentation of that target ion to produce a range of daughter ions. One (or more) of these fragment daughter ions can be selected for quantification purposes. Only compounds that meet both these criteria, i.e. specific parent ion and specific daughter ions corresponding to the mass of the molecule of interest are isolated within the mass spectrometer. By ignoring all other ions that flow into the mass spectrometer the experiment gains sensitivity, whilst maintaining exquisite accuracy.
What type of samples can be analysed?
Essentially any compound that can be ionized and then fragmented can be used for MRM analysis.The most typical examples being the measurement of proteins and peptides, metabolites, or drugs in complex mixtures such as plasma and serum. Quantification of proteins is possible through the MRM experiment on digested peptides that correspond to the protein of interest. This makes MRM analysis a common choice for biomarker analysis and for detection and quantification. As the name suggests more than one MRM can be monitored per analysis allowing detection of multiple compounds in the same experiment.
How sensitive is MRM?
This is a highly sensitive technique. It is possible to detect peptide compounds not normally seen by a typical MS approach. For example peptides can be accurately quantitated at femtomole concentration. In complex samples such us plasma, peptides can be quantitated at μg/ml concentration.
Is MRM compatible with absolute or relative quantitation?
This technique can provide results for either type of quantitation. Relative quantitation is generally straightforward but for absolute quantitation appropriate standards are required.
Is method development required?
Yes. Each project is specific and requires considerable amounts of operator time to develop and run the MRM analysis. For absolute quantitation a specific peptide is quantitated against a spiked internal standard (e.g. a synthetic stable isotope labeled peptide standard) to yield a measure of protein concentration. For complex mixtures some sample clean up may be required with significant chromatographic separation. For plasma and serum extra preparation work is required and additional fees will be charged.
APPLICATION EXAMPLES
Protein Quantification MRM
Reference for article on MRM of peptides to determine relative protein quantification: Ravenscroft G., Colley S. M. J., Walker K. R., Clement S., Bringans S. D., Lipscombe R. L., Fabian V. A., Laing N. G. and Nowak K. J. Expression of cardiac α-actin spares extraocular muscles in skeletal muscle α-actin diseases - determination of cardiac α-actin by MRM mass spectrometry. Neuromuscular Disorders, 18, 953-958.
This article describes the development and implementation of an MRM based method to determine relative quantities of two proteins (cardiac and skeletal actin) which differ in only 4 residues out of 375 in their sequences, i.e. they are 99% homolgous. The proteins were processed together and specific MRMs developed that would detect the presence of each form sensitively and specifically. Only relative quantification was required for this application.
Peptide/Lipopeptide MRM
We have developed an MRM application to detect the presence of a 9 residue peptide (mass 1023.8) and its lipidated form (mass 1899.8). Pure standards of each were supplied allowing optimisation of the mass spectrometric conditions specific to each compound. Once MRM transitions were decided then calibration curves were derived for injections of known amounts of each. This allowed quantification of the amounts of these peptides present in the samples to be analysed. The solutions these samples were in was not complex and hence a direct injection approach was applicable with no chromatographic separation before mass spectrometric analysis. Due to the large sample number and high throughput requirements this MRM experiment was performed using an Agilent 1100 system with a flow rate of 200 microL/min into the Turbo-V source of the 4000 Q TRAP. This is less sensitive than the nanospray interface but more than adequate for the application as approximately 10 ng or low pmole amounts were accurately quantifiable with a sample run time of 2 min.
Delivery
2-4 weeks
(Note: Costs and time are project specific)
The proteome is analysed by LC-MALDI: a combination of extensive chromatography separation and mass spectrometry.
Pure protein sample is digested and run through an extended 1- or 2-D LC gradient, and the eluent spotted directly onto a plate for MALDI-TOF/TOF analysis. MS spectra are analysed to identify proteins of interest.
Outcomes of this analysis are dependent on the quantity of protein present, and for 1D-LC will range from identifying only the major proteins, through to 100s of ID's. If the sample is complex (e.g whole cell lysate) more sophisticated 2D-LC is used. The sample is first separated by strong cation exchanged chromatography, followed by reverse-phase chromatography and analysed by MALDI-TOF/TOF. 2D-LC/MS/MS can identify over 1000 proteins in one experiment.
What amount of protein is required?
1D-LC: 10-20μg of pure protein
2D-LC: 100-200μg of pure protein; however if sample requires additional cleanup we recommend 200-500μg.
What type of sample can be analysed?
Essentially any protein sample such as cell lysate, whole tissue, secreted proteins, but not radioactive samples.
#Note: secreted samples must be in serum/plasma free media.
What is the maximum volume of sample?
The sample volume must be less than 250µl.
Can I use protease inhibitors?
Yes, protease inhibitors will minimise protein degradation.
Is it important that your proteome that you are looking at is in currently available databases?
Yes, proteome mapping analysis works best on samples from a known genome.
Where genomes are partially known the normal mapping approach will give limited results, however, Proteomics International's advanced de novo sequencing techniques can be applied; please contact us for a quote.
Delivery
20 working days
Phosphorylation detection by Mass Spectrometry:
For phosphorylation detection we use an LC/MS/MS system (4000Q-Trap [Applied Biosystems]).
Our approach is first to perform protein identification by tandem MS (as per our standard service). This enables confirmation of protein identity prior to detection of the phosphorylated peptides.
How much protein do I need?
We recommend protein bands or spots are of sufficient concentration to be detected by Coomassie G250 staining. Two bands are required to enable the initial protein identification, although less material can be used for that process. Larger amounts of material increase the probability of achieving a successful result.
General Conditions:
Proteomics International Pty Ltd (ABN 78 096 013 456). All prices are in USD. The provision of viable/correct samples are the client's responsibility. Please supply a purchase order number to accept quote. All quotations are valid for 1 month and are subject to PI's standard terms and conditions, available at www.proteomics.com.au.
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