MASCOTNixon Mendez

Department of Bioinformatics

Basics

Simple MS – molecular weight of peptide mixture.

MS/MS (Tandem MS) – sequence structural information by

recording the fragment ion spectrum of peptide.

BASICS

Tandem MS

Mass spectrometry

PURPOSE OF MS

Elemental composition.

Masses of particles of molecules.

Identify unknown compounds.

Isotopic Composition.

INTRODUCTION

Mascot is a software package from Matrix Science

(www.matrixscience.com) that interprets mass spectral data into

protein identities.

It uses mass spectrometry data to identify proteins from primary

sequence databases.

INTRODUCTION

The experimental mass values are then compared with calculated

peptide mass by applying cleavage rules to the entries in a

comprehensive primary sequence database.

If unknown protein is present, we will get precise entry otherwise pull

out those entries which exhibit the closest homology(related species).

Database

Eg. Protein databases -

Non-redundant NCBI,

Swiss-Prot,

IPI, etc.

Peak Lists

In silico digest

820.7

842.5

1012.6

1296.6

1555.7

……...

Algorithm

compares peak

lists

Gel separation – 1D or 2D

Excise

Spot

Trypsin

Digest

Protein Peptides699.0 1159.2 1619.4 2079.6 2539.8 3000.0

Mass (m/z)

1.6E+4

0

10

20

30

40

50

60

70

80

90

100

% Intensity

4700 Reflector Spec #1 MC=>TR[BP = 1479.9, 15779]

1479.8824

1439.8967

1567.8276

1163.7000

2045.1273

927.5582

1881.0223

1724.9272

1305.7888

1730.7723

1399.7751

1249.6954

1895.0386

1283.7881

1433.8074

1554.7437

1640.0277

841.5205

2555.2903

1763.7820

1687.8691

2262.0557

1516.7135

1014.6827

1590.8619

1081.5479

1121.5520

2458.3052

1195.6243

789.5378

898.5428

2493.3501

Mass spectrum (MS)

Peak List820.7

842.5

1012.6

1296.6

1555.7

……...

Reports Protein

Identification

Database searching

Algorithm used..

• Program MASCOT is based on the MOWSE algorithm; this program also

evaluates a possibility of random matching of experimental and

theoretical peptide masses.

• The Algorithm MOWSE (Molecular Weight Search) is more selective and

sensitive.

Two Mascot Choices

Matrix Sciences offers two choice for users:

A free, open access web-based system for occasional (1-10) queries.

A locally installed version for heavy use or highthroughput MS (100’s

queries/day)

MASCOT Home Page

MASCOT SEARCH STRATEGIES

Mascot has three main search modes:

Peptide Mass Fingerprint(based on a list of peptide mass values).

Sequence Query (based on one or more peptide mass values

associated with information such as partial sequence information).

MS/MS Ion Search (based on raw MS/MS data from one or more

peptides).

PEPTIDE MASS FINGERPRINT

• It is possible to identify the protein from available mass spectrum of the peptide mixture

resulting from the digestion of a protein by specific enzyme.

• This method is useful for identifications of protein with already known sequences.

• Requires enzymes of great specificity.

• Mascot looks for the highest scoring set of peptide matches which are within a contiguous

stretch of sequence less than or equal to the specified protein molecular weight.

SEQUENCE QUERY

• The sequence query, in which one or more peptide molecular masses are combined with sequence,

composition and fragment ion data

• It is potentially the most powerful search.

• The source of the information is MS/MS spectrum.

• The sequence query mode of Mascot supports both standard and error tolerant sequence tags.

MS/MS IONS SEARCH

• The MS/MS ions search accepts data in the form of peak lists containing mass and intensity

pairs.

• The high level of specificity of an MS/MS ions search means that it is not essential to choose

an enzyme.

• Obtaining matches to a number of peptides from a single protein provides a very high level of

confidence that the result is correct.

PEPTIDE MASS FINGERPRINT

Parameters used in database

searching

Database searched

Taxonomy

Enzyme

Missed cleavages

Fixed versus variable modifications (PTMs)

SCORING SCHEMES

PROBABILITY BASED SCORING

Mascot incorporates a probability based implementation of the Mowse

algorithm

The total score is the absolute probability that the observed match is

a random event.

Advantages :

Different types of matching (peptide masses and fragment ions) can

be combined in a single search.

Scores from different searches and on different databases can be

compared.

Search parameters can be optimised more readily by iteration.

For this reason,

We report scores as -10*LOG10(P), where P is the absolute probability.

Probability of 10-20 thus becomes a score of 200.

Result

The best result is obtained for PML_HUMAN having a score of 194, we

can confirm it by referring the graph where there is one hit and 194

lies in the significant region having a threshold score of 70 so the

chances of randomness is reduced.

The hits below the threshold region are insignificant.

Result

MS/MS IONS SEARCH

Parameters

Additional parameters within each query are optional, and can be used to

specify:

TITLE for spectrum identification

CHARGE state of the precursor peptide

TOL peptide tolerance

TOLU peptide tolerance units

SEQ for a sequence qualifier (multiple SEQ qualifiers are allowed)

COMP for a composition qualifier (only one COMP qualifier is allowed)

TAG for a sequence tag (multiple TAG qualifiers are allowed)

Result

There is only one hit having a score of 180 that falls in the significant

region.

In MS/MS Ion search the best result is taken by number of queries

matched and the score should be highlighted in bold & red.

Sequence Query

SEQUENCE QUERY

Result

Result

Here we obtain 3 hits for the score 76, which fall in the significant

region.

So, here the best match is selected by the numbers of the queries

matched.

LYSCO_PHACO is the best match for this result.

Thank You