Pick ‘n Post Services – Protein Analysis in 10 Minutes

PICK ‘n POST are fast and high-quality protein analysis services offered to research scientists in biotech, pharma and academia.

Your samples are easily prepared, packed and conveniently shipped by express courier using the PICK ‘n POST kit. A detailed result report is returned to you within 2 to 8 days depending on the service selected.

Choose from these easy to use services:

1. Protein Identification of bands & spots from 1D/2D SDS PAGE gels.
2. N-terminal Edman Sequencing of proteins and peptides.
3. Amino Acid Analysis of proteins, peptides & free Amino Acids.
4. Molecular Weight Determination of proteins and peptides.

All services are conducted at a fixed fee-per-service. We pick up your samples in your lab as part of the Pick ‘n Post service. The Pick ‘n Post™ kits are supplied free of charge and contain all the information and materials needed for easy and secure shipment of samples to Alphalyse.

What buffer should I use for MALDI mass spectrometry analysis of my protein sample?

Question:

What buffer should I use for MALDI Mass Spectrometry analysis of my protein sample?

Answer:

Although MALDI is more tolerant to contamination than other MS methods, the salts and detergents commonly used in biomolecular science can still interfere with either crystallisation or ionisation or both. Particular attention should therefore be paid to using sample purification and storage solvents which do not utilise buffers, salts or detergents such as SDS.

It is best to remove buffer salts and detergents (e.g. by dialysis) prior to analysis and to dissolve the sample in a suitable solvent (e.g. 0.1% TFA/water) which will not degrade the spectrum. If there is too much salt in a sample, the salt signal intensity is so large that it effectively suppresses out the sample signal, giving no sample spectrum. In cases where it is not possible to remove these contaminants the sample should be in a higher concentration. It may then be possible to dilute the sample to the point where the contaminants will have little effect on the spectrum.

One would normally like to have any sample in a volatile buffer not containing any salts and detergents. Examples of volatile buffer are ammonium acetate, acetonitrile, ammonium formate, TFA (trifluoroacetic acid) or formic acid in water.

Buffers not compatible with MALDI Mass Spec analysis of proteins include:

• TRIS
• PBS

 

Buffer components that will not work with Mass Spec analysis include:

• Tween 80
• Tween 20
• Triton X
• Glycerol
• DMSO
• SDS
• Sodium Azide

Learn more about all of our protein analysis and characterization services on our website: www.alphalyse.com

I just got my protein identification report. What does the mascot score mean?

Question:

I just got my protein identification report. What does the mascot score mean?

Answer:

The protein ID report contains the protein(s) that have been identified positively by their score in the Mascot database search.

When we run MALDI mass spectroscopy and MS/MS the data is combined and searched in the database. The hits we get from the database are based on several proteins that are sequenced. Mascot shows which proteins could match the protein or proteins in each sample based on the MS and MS/MS data. When we determine which hit is the correct, we compare the information on the sample submission form with the hit from the database.

We also look at the mascot score and the individual ion-score for each peptide. The mascot score has to be inside the 95% confidence level (Mascot scores higher than 90 or a single ion-score higher than 20). Much of the analysis is based on the mass of the peptides that are identified and using bioinformatics these are compared to the masses of sequences in the NCBI database. What you need to keep in mind with the Total Mascot Score is that this number will go up with time. At the moment there are >10 million protein entries in the public databases. A few years ago there were only 2.5 million and at that time a Total Mascot Score of 62 gave a significance of >95%. I am sure that at some time in the future the Total Mascot Score needed for significance will be above 100.

You will also note that in our reports the peptides listed in bold (under the section “Peptides used for identification”) were individually sequenced by MS/MS. Ion scores above 20 are significant and above 30 are considered certain. The mascot score is described in details at the Matrix Science website (http://www.matrixscience.com/). It is a logarithmic scoring.

Want to find out more about protein identification of bands and spots from 1D and 2D SDS PAGE gels using mass spec? Just follow this link to our PICK ‘n POST protein identification website .

The turn-around time for our Pick ‘n Post protein identification service is 2-4 days regardless of the number of samples submitted.

How do I prepare a sample for N- and C-terminal sequencing by MALDI In-Source-Decay (ISD)?

Question:

I am interested in your N and C-terminal sequencing using MALDI ISD. How do I prepare the sample for analysis?

Answer:

MALDI ISD is used for confirmation of the N- and C-term of know proteins therefore you would need to supply us with the sequence of the protein you are working with so that we can confirm the sequence of the two termini. We require the samples to be in liquid form with a purity >80%. We need 20 ug of material to do the analysis if you can provide more this would be ideal. The more material we receive the better the quality of the results will be.

When you submit your samples you should list what is in the buffer you are using. Please let us know if there is glycerol in the sample as we need to remove this before placing the sample in the mass spec. The sample should be purified by reversed-phase HPLC.

You should note that the N- and C-terminal sequencing by MALDI ISD is done simultaneously on each sample.

Read more about N- and C-terminal sequencing using MALDI In Source Decay (ISD) in our application note, and about the Alphalyse N- and C-terminal sequencing service

Identification of novel proteins not present in public databases

Question:

My protein was analyzed using Alphalyse’s Pick ‘n Post protein identification service. The sample gave very good MS data, but was not identified because it is a novel protein not yet present in the NCBI database. Can you provide me the raw MS peptide fingerprint spectra and MS/MS peptide sequence data for additional database searching?

Answer:

For unidentified proteins with good MS data, Alphalyse can provide Mascot Generic Format files (MGF files) containing the MS data that was used for the Mascot database search. The MGF files contain the peptide masses and the peptide fragment masses obtained for each sample in a format that can be uploaded to the Mascot database search software.

The MGF file will enable you to repeat the database search:

A) on your own in-house Mascot search engine with proprietary databases,

B) on the public Mascot server http://www.matrixscience.com/ when more protein sequences are added in the future, and

C) on other database search programs using different search algorithms.

Please contact Alphalyse at info@alphalyse.com for a price quotation on the MGF file conversion for your samples, and to obtain a small tutorial on how to perform the MGF Mascot database searches. Learn more about protein identification using mass spectrometry.

Databases and organisms available for mass spec protein identification service

Question:

I would like to identify proteins from an organism where the genome has just been sequenced.  How many proteins are available in the database you use, and from what organisms?

Answer:

The database used for Pick ‘n Post protein identification is the public nr database (nrdb) from NCBI. The database is a non-redundant (nr) compilation of all known protein sequences from GenBank CDS translations + PDB + SwissProt + PIR + PRF. The database is constantly updated with new sequences and downloaded regularly to our in-house Mascot server. Currently, the database contains more than 9 million protein sequences.

To find out how many protein sequences are included for your organism, go to the NCBI Entrez website and select the specific organism.

The NCBI Entrez Taxonomy Homepage:

http://www.ncbi.nlm.nih.gov/sites/entrez?db=taxonomy

Or search the database by organism name in Protein search:

http://www.ncbi.nlm.nih.gov/sites/entrez?db=Protein&itool=toolbar

Useful Protein Molecular Weight Calculator

Protein molecular weight calculator

The rule of thumb for conversion of microgram amounts into picomole amounts at different protein molecular weights and peptide molecular weights is:

 1000/Mw of protein in kDa = pmol/ug 

Protein size kDa	Microgram	Picomole	Microgram	Picomole
1	1	1000	1	1000
10	10	1000	1	100
20	20	1000	1	50
50	50	1000	1	20
100	100	1000	1	10
150	150	1000	1	6.7

Useful Links: 

Protein molar calculator and formulas:

http://www.promega.com/biomath/calc08.htm

Coding capacity of DNA, conversion of DNA base pairs to approximate protein Mw:

http://www.promega.com/biomath/calc09.htm

Compute pI/Mw tool from protein amino acid sequence or database accession number:

http://www.expasy.ch/tools/pi_tool.html

Protein Mw determination by mass spectrometry:

http://www.pick-n-post.com/default.asp?ID=50010300070

Common protein contaminants observed in mass spectrometric protein identification

Questions:

1. Is the keratin identified by the Pick ‘n Post protein ID service in my 1D gel band a contamination or a relevant human protein from the sample?
2. How is it possible that only Keratin was identified in the sample although the protein band was clearly visible?
3. Does this mean that keratin was the only protein in the sample or is the identification of other proteins impossible when even small amounts of keratin are present?

• The size of my protein and band cut from gel was 50-55kDa and Keratin is 66kDa, so how did the Keratin get into sample?
• Is there even a smallest possibility that something happened in your lab?

These are just few things that come to my mind as I’m thinking what might have gone wrong. It would be nice if you could help me with these concerns, before I’ll start the whole process once again.

Answers:

When keratin is identified in a gel band it might be a real protein purified from the cells, but more frequently it is a contamination that occurred somewhere during protein purification and sample processing. Keratin is present in all dust in the lab from dead skin cells from humans and small pieces from your woollen sweater!

If the keratin contamination occurred prior to the gel electrophoresis, the keratin is observed as protein bands in the gel around 55 kDa and 65 kDa. This keratin is identified with a high score and good sequence coverage, and often several keratin types are identified. Your gel band was observed at Mw 55 kDa, so either this is a contamination that happened before the electrophoresis, or it was purified from the cells.

Keratin contamination can also occur after the electrophoresis during gel staining, or during gel scanning and spot excision if dust comes into contact with the sample. In such cases, the keratin amount is usually lower and only a few keratin peptides are observed in the mass spectra. The main protein component in the gel band can still be identified.

At Alphalyse we take extreme care to work in a clean dust-free environment. All samples are handled in 96-well plates together with quality control standards, and contamination from our lab is almost never observed.

The best advice to avoid keratin contamination is to avoid dust in lab in general, and always work with gloves. Electrophoresis and staining equipment should be cleaned and free of dust, and the lids on pipette tips should always be closed.

Additional Refrences:

Common protein and keratin contaminants observed in proteomics experiments are given in the references below.

The Common Repository of Adventitious Proteins, cRAP, is a list of proteins commonly found in proteomics experiments that are present either by accident or through contamination of protein samples.

keratin 1 (SWISS-PROT: P04264); similar to keratin 1 (ENSP00000301445); keratin 2a (SWISS-PROT:P35508); similar to keratin 2a (ENSP00000252247); keratin 5 (ENSP00000252242); keratin, type II cytoskeletal 6F (SWISS-PROT:P48669); keratin 9 (ENSP00000246662); similar to keratin, type I cytoskeletal 10 (SWISS-PROT: P13645); keratin 10 (TREMBL: Q14664); keratin 14 (SWISS-PROT: P02533); keratin 16 (ENSP00000301653).

Were observed  in publication:

Large-Scale Proteomic Analysis of the Human Spliceosome. Juri Rappsilber, Ursula Ryder, Angus I. Lamond, and Matthias Mann. Genome Res. 2002. 12: 1231-1245 http://genome.cshlp.org/content/12/8/1231.full

Common Peptide Contaminants Observed by Nanoelectrospray MS in Low Level Sequencing of Gel-Separated Proteins. Jens S. Andersen, Bernhard Küster, Alexandre Podtelejnikov, Ejvind Mørtz, and Matthias Mann.

with a list of contaminating peptides (peptide mass, sequence, Y-ion fragment masses) from keratins, and trypsin autolysis peptides from Roche bovine trypsin, and Promega modified trypsin. www.pil.sdu.dk/files/ContaTableASMS1999.doc

How do I assess the stability of a protein drug substance and formulated drug product?

After protein purification, an important issue is the stability of a protein drug substance and formulated drug product. Stability assays of protein vaccines formulated with alum adjuvants are not straightforward to perform because most standard analytical methods for protein characterization cannot be easily applied to proteins immobilized on alum.

Alphalyse performed a stability assay on a protein vaccine. Vaccine vials were incubated at the normal storage temperature at 4 °C and at 37 °C in an accelerated stability study. Samples were taken at different storage intervals for stability measurements. For analysis of protein degradation products, the protein was eluted from the alum hydroxide by treatment with SDS-PAGE sample buffer. The samples were analyzed by electrophoresis, and the gels were stained with a sensitive silver staining method compatible with MS analysis.

The figure below shows a silver-stained gel of the drug product stored for nine months at 37 °C in the accelerated stability study. Degradation protein fragment bands were cut out from the silver-stained gel and analyzed by in-gel trypsin digestion and MS peptide mapping. The protein sequence coverage map shows peptide maps obtained for the individual protein fragments. Those peptide maps confirm that the proteins are drug substance degradation products. The sequence coverage maps show that bands 1 and 2 are protein fragments missing the C-terminal region, band 3 and 4 are also missing the N-terminal region, and band 5 and 6 found around 6 kDa in the gel contain only the middle part of the sequence. Thus, the combination of SDS-PAGE and MS peptide mapping again provided very valuable information (about protein degradation patterns, in this case) that is not easily obtained by other analytical techniques.

 

Stability analysis of alum formulated protein stored at 37oC for 9 months.

Figure: Stability analysis of alum-formulated protein stored at 37 °C for nine months. The protein was eluted from the alum, and 10 μg were analyzed by 1D SDS-PAGE. The break-down products (Bands 1–6) were cut out from the silver-stained gel together with intact protein (Band 0) and analyzed by MALDI MS peptide mapping. Peptide masses obtained from the degradation products were correlated to the protein sequence using GPMAW software from Lighthouse Data (www.gpmaw.com), and the results are shown in the protein sequence coverage map.

More about Protein Stability Assays here

How to optimize electroblotting conditions onto PVDF membrane for N-terminal Edman sequencing

How do I optimize electroblotting conditions onto PVDF membrane for N-terminal Edman sequencing? Our 12 kDa protein separates well on 1D SDS PAGE but does not appear on the PVDF blot for N-terminal sequencing. We are using the NuPAGE Transfer buffer with 20% MeOH from Invitrogen. What can we do to optimize the blotting conditions?

Answer: 

The blotting buffer generally works well for most proteins. However, some proteins may show poor electroblotting efficiency, and the choice of PVDF membrane, blotting buffer and blotting conditions should be optimized. During optimization it is an advantage to stain the gel after blotting and to use 2 layers of PVDF. Some large proteins (above 80 kDa) may be difficult to get out of the gel, and it can help to add 0.1% SDS to the buffer since SDS increases the mobility of the proteins. The same effect can be obtained by omitting MeOH from the buffer, because MeOH strips SDS from the protein.

Some small proteins (below 15 kDa) may move too quickly out of the gel and through to the first PVDF membrane. In that case, SDS should not be used and the MeOH concentration increased to 20%. Also the gel can be pre-soaked in blotting buffer for 5-10 mins before blotting. Choice of blotting buffers with a neutral pH (Tris-Glycine buffers), may be useful for very basic proteins with high isoelectric points. Basic proteins may be positively charged and the PVDF membrane should be placed on the other or on both sides of the gel. Glycine-containing buffers will give high glycine yield in the first Edman cycle, and the PVDF membrane should be washed extensively after staining. Use this PVDF transfer protocol for N-terminal Edman Sequencing