Welcome

Database search is mainly used for protein identification, but it can also assist with characterization of recombinant proteins.

In this month's featured publication, the authors elucidate histone modifications and truncations. If you have a recent publication that you would like us to consider for an upcoming Newsletter, please send us a PDF or a URL.

Mascot tip of the month describes how to submit a search at the command line when the peak list is too big for the web server to handle.

Please have a read and feel free to contact us if you have any comments or questions.

Assuring protein quality with database searching

Quality assurance of recombinant proteins is a tough problem, and many analytical techniques are required to verify the primary sequence, modifications, crosslinking, and freedom from undesirable contaminants of the protein product. Although database search was designed for the identification of unknown proteins, it also has a role to play in a QA environment, but we must emphasise that it is for research use only. We are not aware of any regulatory approval for its use in connection with therapeutics (yet!).

Try to avoid searching a sequence database with just one entry, or a very small database where the entries are variants of the same protein. There are no meaningful statistical measures of significance in such searches, making it hard to decide whether a low scoring match is correct or simply a chance peptide molecular mass match. Including common contaminants and the host cell proteome in the search will help give some confidence in the matches even if there are too few for target-decoy validation.

An error tolerant search is ideal for picking up peptides modified by artefacts, such as oxidation or over-alkylation, not to mention non-specific cleavage and the occasional post-translational modification or SNP. For additional tips relevant to QA-type searches, read this recent blog article

Featured publication using Mascot

Here we highlight a recent interesting and important publication that employs Mascot for protein identification, quantitation, or characterization. If you would like one of your papers highlighted here please send us a PDF or a URL.

Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns

Andrey Tvardovskiy, Krzysztof Wrzesinski, Simone Sidoli, Stephen J. Fey, Adelina Rogowska-Wrzesinska and Ole N. Jensen

Molecular & Cellular Proteomics (2015), 14, 3142-3153

Post translational modifications of histones play a major role in regulating chromatin functionality and the concomitant DNA processes. It is thought that not only "classical" histone PTMs, such as methylation, acetylation, and phosphorylation effect the function, but also the proteolytic cleavage of the N-termini known as "histone clipping".

This paper takes on the challenging characterization of the co-existing histone modifications and reveals the relationship between histone clipping and covalent histone PTMs. Using immunoblotting as well as top- and middle-down mass spectrometry methods, the authors showed the presence of clipped histones and the co-existence of various PTMs for H3 histones.

They found that histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A. They mapped 212 unique combinatorial PTMs on intact H3 N-terminal tails and 55 combinatorial PTMs on two different clipped H3 N-terminal tails.

Mascot tip of the month

If your Mascot Server is on Windows and the web server is IIS then there is a limit of 4 GB on the size of the peak list submitted for the search. You can submit larger files from Mascot Daemon by using Apache as the web server, but working with very large files can still be difficult due to time-outs. If you only search very large files occasionally, another option is to bypass the web server and submit the search at the command line:

1. In the Options section of mascot.dat, change the argument for SaveEveryLastQueryAsc from 0 to 1 and save
2. From a browser, submit a search using exactly the same search parameters as you intend to use for the large file, but choose some small text file that isn't a valid peak list
3. The search will fail. In today's data directory, there will be a new file with the extension *.inp, e.g. F001234.dat.inp
4. Open this *.inp file in a text editor
5. Copy all of the lines before the first line of the dummy text file and save as (say) head.txt
6. Copy all of the lines after the last line of the dummy text file and save as (say) foot.txt
7. Concatenate the header and footer with your big MGF using Windows copy:
   copy head.txt+big.mgf+foot.txt input.txt
    
   or Linux cat:
   cat head.txt big.mgf foot.txt > input.txt
    
   
8. Use input.txt to run the search at a command prompt, e.g. Windows:
   cd \inetpub\mascot\cgi
   nph-mascot.exe 1 < input.txt
    
   or Linux:
   cd /usr/local/mascot/cgi
   ./nph-mascot.exe 1 < input.txt
    
   

About Matrix Science

Matrix Science is a provider of bioinformatics tools to proteomics researchers and scientists, enabling the rapid, confident identification and quantitation of proteins. Mascot software products fully support data from mass spectrometry instruments made by Agilent, Bruker, Sciex, Shimadzu, Thermo Scientific, and Waters.

Please contact us or one of our marketing partners for more information on how you can power your proteomics with Mascot.

Matrix Science Ltd, 64 Baker Street, London W1U 7GB, UK
T +44 (0)20 7486 1050  F +44 (0)20 7224 1344  E info@matrixscience.com