MS Uni OS


For a description of a appropriate data aquisition and workflow see --> HERE

How the program works with tutorial data can be tested --> HERE. The corresponding MSMS Search of the same data is ready for download--> HERE

If any help or information is needed, do not hesitate to come in --> CONTACT

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Sequence
accession
number(s)

Search for
entry name via
uniprot.org
1:

2:

3:
located
in database

Homomers allow self aggregation (homomer formation)

Enzyme Allow up to missed cleavages

Fixed
modifications

Display all modifications   
Variable
modifications

crosslinker chemical: peptide:
max nrs of
crosslinked
peptides
password
(if more than 5)
tolerance MS  ±  MS/MS  ± 
crosslink's charge use       charge state, determined by deconvolution
use mass values monoisotopic    averaged
Data format Data file
append or use
single mass(es)
with m/z
ETD fragments analyse most intense ETD MSMS-fragments

Show results with incomplete ETD fragmentation pattern
Mascot-search with   MS2 ETD data MS3 CID data (not with mgf files)



Charge state

This charge state value(s) is/are applied for the crosslinked peptides or, in other words, at the MS1-level.The charge state has to be resolved in order to calculate the precise molecular weight.

Normally this value can be determined by a deconvolution software analysing the raw data. This software based determination is strictly dependend on the quality of the raw data.But the molecular weigth of crosslinked peptides is often above 3 kDa and the charge state is often higher than 4. This combination reaches the resolving power of the MS and therefore the charge state is not or wrong calculated.

Here you have the possibility to influence the charge state

Examples:

Single mass(es)

Here you can enter manually one or more m/z values, which have to be included in the subsequently analysis. This helps to analyses peaks (m/z), which are not included in the scan list or compound list. The different values have to be separated by semicolon and the charge state have to be specified.

Because of missing a lot of further information to this m/z's (i.e. ETD spectra), only simple version of the analysis (only MS) can be done

Single masses can be used without any further MS-data

Mass calculation mode

see a very good information at matrixscience

Enzyme

Select the enzyme/method which was used to generate peptides.

Be very carefully by using "none": This means, that the protein sequence is cut without any specificity. This results in thousand or more peptides

NoCleave means, that the protein sequence is used intact (with the exception of cutting the fMet at the beginning of the protein)


Allow up to

Setting the number of allowed missed cleavage sites. s

If experience shows that your digest mixtures usually include some partials, you should choose a setting of 1, or maybe 2 missed cleavage sites. Don't specify a higher number without good reason, because each additional level of missed cleavages increases dramatically the number of calculated peptide masses.

MS2 ETD

During the ETD fragmentation process the dominant fragmentation process in C-C containing peptides is the break of the C-C linkage.
But in minor degree the peptides itselves undergo the normal backbone fragmentation. This fragmentation pattern can be used to determine the corresponding peptide sequence by searching a database. But therefore, the molecular weight of the precursor ion has to be changed, because in the case of C-C linked peptides the standard used precursor mass corresponds to the crosslinked peptides,but the ETD fragmentation pattern fits the single peptides within the complex.

This program connects the ETD fragmentation pattern with the masses (as precursor mass) of all spectulated peptide within the complex.

As consequence in the database search results you will see several, so called "MS2(ETD)" pseudo peptides.




MS3 CID

The sequence of the peptides, which has been released from the crosslinked complex by ETD, can be confirmed by an additional isolation and fragmentation step.
This has to be done as a MS3 experiment with the help of CID fragmentation. These data (if present in the MS data file) will be collected, reformated as MS2 data and subsequently used for a database identification search. The corresponding spectra are labelled as "MS3 CID" as seen in the result file

Chemical crosslinker

Here you can enter the chemical formula of a crosslinker, which has an active -SH linkage group. This group has to be addressed separately, i.e. C12H22OSH.

Subsequently the program calculates the molecular weight of the crosslinker and include the data in the identification process.

Prerequisite for the positive identification is the stability of the crosslinker during the electron transfer (ETD) in the fragmentation process


Peptide crosslinker

A peptide sequence in upper case letters (allowed are the 20 standard amino acid single letter abbreviation). At least one cysteine (C) has to be located in the sequence in order to realize an cysteine linkage between the peptide and any of the other peptides, derivate from the given protein sequence.

The given peptide sequence is not included in the digestion procedure although a recognition site for the selected enzyme is present

Non of the modifications will be applied to crosslinker peptide sequence

Sequence accession number(s)

Enter the accession number of the protein, which has to be analyzed. If you don't know this number, you can use the given links to find the database entry (=accession number) in the corresponding databases.

Advantage of using SwissProt or UniProt, is the availability of information about already identified or postulated C-C linkages.


located in database

Select the database, which exactly fits to the given accession number

Show incomplete results too

In some cases the MS-data (mass of the linked peptides) are fitting very well

But inspecting the ETD-data (which represent the mass of each of the single peptides) one peak (= one peptide) is missing.

By activating this option these incomplete data are also summarised with the intension, that the raw data will be rechecked by the operator itself.

data format

Several input formats can be chosen:

  1. MGM
    this mascot generic file format contains the MS and MS2 data in a text based manner. It' very common to export MS data into this format.The disadvantage is the limited capacity for information: Neither MS3 data nor charge state values for the MS2 data can be stored. Thereforethe usage of the CID MS3 data, which are a part of this analysis, is omitted.
  2. mzML
    This xml based data format is one of the modern standard of storing MS data (see HUPO-PSI ). Some vendors of MS-software implemented the export of all MS-data types to this format, but with some specific "interpretation" of rules.
  3. Bruker DA xml:
    Bruker's data analysis allows the export of so called "compound list". These list contains structured MS-data, unique for this vendor. For generation of this compound list load the the raw data file (analysis.yep) into Bruker data analysis (at least version 4.0), create compounds, deconvolute them and subsequently export the list as xml.
  4. Thermo raw data:
    The usage of the raw data file generated from the OrbiTrap machines is possible. Simply load this file in the upload formular. The file will be read, a peak peaking filter applied and the scans analyzed.

Be sure, that the data format fits perfectly to the file type, which has to be selected too.

Fixed or variable modifications

Select any known or suspected modifications.

Two types of modification are supported:

The best advice is to use variable modifications sparingly; never select a large number "just in case".

Several overlapping combination of fixed or variable modifications were excluded in order to clear the situation



The 'Show all mods.' checkbox switches between a short list of the most common modifications and a complete list of all available modifications.

ETD fragments

The crosslinked peptides are broken apart by ETD. This reaction occurs mostly dominant.

Here you choose the number of dominant peaks, which have to be included in the C-C identification process.

This value has to be greater than the expected number of peptides in the most complex aggregation

Homomers

By activating this option, self aggregation of the generated peptides is allowed.
In the case of homo-multimers it is not necessaryto enter the protein accession number several times.

The degree of self aggregation is not limited, so the same peptide can be found crosslinked with itself several times (if enough cyteins are located in the sequence and the "maximal numbers of peptids" which form the aggregation is set high enough)

Enter the tolerance value, which is used while comparing deduced ( from the amino acid sequence) and experimental (measured by the mass spectrometer)mass data. Select an appropriate unit

MS tolerance

This tolerance value is applied for the crosslinked peptides or, in other words, at the MS1-level


MS/MS tolerance

This tolerance value is applied for the single peptides which has been separated by ETD or, in other words, at the MS2-level (ETD-spectra)

max nrs of crosslinked peptides

Enter the expected maximum number of peptides, which will be organized in the multimer with the highest complexity

This value is the most critical point in the analysis, because the increase results in an dramatic length of the running time and processor usage.

In order to avoid blockage of the server, the number is limited to 5

If it is necessary to increase this number, please make contact via EMail to receive an password for abolishment of this limitation