Computational Methods for Mass Spectrometry Proteomics

by Ingvar Eidhammer (University of Bergen, Norway ); Kristian Flikka (University of Bergen, Norway ); Lennart Martens (European Bioinformatics Institute (EBI), Cambridge, UK ); Svein-Ole Mikalsen (The Norwegian Radium Hospital, Oslo, Norway )

• ISBN13: 9780470724293
• ISBN10: 0470724293
• Format: eBook
• Copyright: 2008-04-01
• Publisher: Wiley-Interscience

Summary

Proteomics is the study of the subsets of proteins present in different parts of an organism and how they change with time and varying conditions. Mass spectrometry is the leading technology used in proteomics, and the field relies heavily on bioinformatics to process and analyze the acquired data. Since recent years have seen tremendous developments in instrumentation and proteomics-related bioinformatics, there is clearly a need for a solid introduction to the crossroads where proteomics and bioinformatics meet.Computational Methods for Mass Spectrometry Proteomics describes the different instruments and methodologies used in proteomics in a unified manner. The authors put an emphasis on the computational methods for the different phases of a proteomics analysis, but the underlying principles in protein chemistry and instrument technology are also described. The book is illustrated by a number of figures and examples, and contains exercises for the reader. Written in an accessible yet rigorous style, it is a valuable reference for both informaticians and biologists.Computational Methods for Mass Spectrometry Proteomics is suited for advanced undergraduate and graduate students of bioinformatics and molecular biology with an interest in proteomics. It also provides a good introduction and reference source for researchers new to proteomics, and for people who come into more peripheral contact with the field.

Table of Contents

Preface

Acknowledgements.

Protein, Proteome, and Proteomics

Primary goals for studying proteomes

Defining the protein.

Protein identity.

Splice variants

Allelic variants - polymorphisms.

Posttranslational modifications

Protein isoforms.

Protein properties - attributes and values.

Molecular mass

Isoelectric point

Hydrophobicity.

Amino acid composition

Posttranslational modifications

Protein sequence databases.

UniProt KnowledgeBase (Swiss-Prot/TrEMBL, PIR

The NCBI non-redundant database.

The International Protein Index (IPI).

Time-instability of sequence databases.

Identification and characterization of proteins.

Top-down and bottom-up proteomics.

Protein digestion into peptides

Two approaches for bottom-up protein analysis by mass spectrometry

MS - Peptide mass fingerprinting.

MS/MS - Tandem MS.

Combination approaches.

Reducing the search space

Instrument calibration and measuring errors.

Calibration

Accuracy and precision.

Exercises

Bibliographic notes

Protein Separation - 2D Gel Electrophoresis

Separation on molecular mass - SDS-PAGE.

Estimating the protein mass

Separation on isoelectric point - IEF

Separation on mass and isoelectric point, 2D

Transferring the proteins from the first to the second dimension.

Visualizing the proteins after separation.

Problems.

Excising the proteins.

2D SDS-PAGE for (complete) proteomics.

Identifying the proteins

Quantification.

Programs for treating and comparing gels.

Comparing results from different experiments - DIGE

Exercises

Bibliographic notes

Protein Digestion

Experimental digestion.

Cleavage specificity.

Trypsin.

Chymotrypsin.

Other considerations for the choice of a protease

Random cleavage

Chemical cleavage.

In-gel digestion.

In silico digestion

Exercises

Bibliographic notes

Peptide Separation - HPLC

High Pressure Liquid Chromatography - HPLC

Stationary phases and separation modes.

Reverse phase chromatography, RP.

Strong cation exchange chromatography, SCX.

Other types of chromatography for proteomics.

Tandem HPLC

Component migration and retention time.

The shape of the peaks.

The width.

Asymmetry.

Resolution.

Chromatography used for protein identification.

Theoretical calculation of the retention time for reverse phase chromatography.

Chromatography used for quantification.

Exercises

Bibliographic notes.

Fundamentals of Mass Spectrometry

The principle of mass spectrometry

Ionization sources.

MALDI - Matrix Assisted Laser Desorption Ionization

ESI - Electrospray Ionization

Other ionization sources.

Mass analyzers.

Isotopic composition of peptides

Estimating the charge

Fractional masses.

Estimating one or two peptides in a peak complex

The raw data

Mass resolution and resolving power

Isotopic resolution

Exercises

Bibliographic notes.

Mass Spectrometry - MALDI-TOF

Time-of-flight analyzers and their resolution.

Time-to-mass converter

Producing spectra.

Ionization statistics

Constructing the peak list.

Noise

Baseline correction.

Smoothing and noise reduction

Peak detection

Example.

Intensity normalization.

Calibration

Peak list preprocessing.

Monoisotoping and deisotoping

Removing spurious peaks

Peak list format.

Automation of MALDI-TOF-MS

Exercises

Bibliographic notes.

Protein Identification and Characterization by MS

The main search procedure

The experimental data

The database - the theoretical data.

Other search parameters

Organization of the database

The peptide mass comparison.

Reasons why experimental masses may not match

Database search and recalibration

The search program MSA (Mass Spectra Analyzer

Aldente.

Score calculation.

Score components

Scoring scheme examples

Identification from a protein mixture.

Statistical significance - the P-value

A priori probability for k matches.

Simulation for determining the P-value.

A simple Mascot search.

Characterization.

Exercises

Bibliographic notes

Tandem MS or MS/MS Analysis

Peptide fragments.

Fragmentation techniques

MS/MS spectrometers.

Analyzers for MS/MS.

Different types of analyzers.

TOF/TOF.

Triple quadrupole (Triple quad

Ion trap (IT).

Fourier Transform Ion Cyclotron Resonance (FT-ICR

Combining quadrupole and Time of flight - Q-TOF

Combining quadrupole and ion trap - Q-TRAP

Combining TOF and Ion trap

Combining Linear ion trap with Orbitrap

Characteristics and performances of some type of analyzers

Overview of the process for MS/MS analysis

Fragment ion masses and residue masses.

Deisotoping and charge state deconvolution.

Precursor treatment.

Precursor mass correction

Estimating the charge state of the precursor

MS3 spectra

Exercises

Bibliographic notes.

Fragmentation Models

Chemical approach.

The mobile proton model, MPM.

Statistical approach.

Constructing the training set(s

Spectral subsets.

Learning (collecting statistics).

Fragmentation Intensity Ratio (FIR).

Linear models.

Use of decision trees

The effect of amino acids on the fragmentation.

Selective fragmentation.

Exercises

Bibliographic notes

Identification and Characterization by MS/MS

Effect of operations (modifications - mutations) on spectra

Comparison including modifications.

Filtering and organization of the database.

Scoring and statistical significance

Exercises

Spectral Comparisons

Constructing a theoretical spectrum

Non-probabilistic scoring.

Number and intensities of matching peaks or intervals

Spectral contrast angle

Cross-correlation

Rank based scoring.

SEQUEST scoring.

Probabilistic scoring.

Bayesian method - SCOPE

Use of log-odds - OLAV.

Log-odds decision trees.

Comparison with modifications.

Zone modification searching

Spectral convolution and spectral alignment.

Exercises

Bibliographic notes

Sequencial Comparison - de novo Sequencing

Spectrum graphs.

A general spectrum graph

Preprocessing

Node scores

Constructing the spectrum graph

The sequencing procedure using spectrum graphs.

Searching the graph.

Scoring the derived sequences against the spectrum

Combined spectra to improve de novo sequencing

Use of two fragmentation techniques.

Exercises

Bibliographic and additional notes

Database Searching for De Novo Sequences

Using general sequence search programs.

The main principle of FASTA and BLAST.

Changing the operation of FASTA/BLAST

Scoring and statistical significance.

Specialized search programs.

OpenSea.

SPIDER.

Peptide sequence tags

A general model for peptide sequence tag search programs

Automatic extraction and scoring of sequence tags

Database search.

Extending the sequence tag hits with flanking amino acids

Scoring the PST matches

Statistical significance.

Comparison by threading.

Use of suffix tree.

Use of deterministic finite automata.

Exercises

Bibliographic notes.

Large-Scale Proteomics

Coverage and complexity

Selecting a representative peptide sample - COFRADIC

Separating peptides into fractions

Producing MS/MS spectra

Spectra filtering.

Classifying good and bad spectra.

Use of the classifier.

Spectrum clustering.

Recognizing sibling spectra

Clustering of sibling spectra

Representative spectra for the groups

De novo sequencing from representative PRM spectra

Searching the database.

LIMS.

Exercises

Bibliograpic notes

Quantitative Mass Spectrometry-Based Proteomics

Defining the quantification task

mRNA and protein quantification

Quantification of peaks.

Normalization

Different methods for quantification

Label-free quantification

Comparing spectra.

MALDI-TOF based methods

SELDI-TOF based methods

LC-MS quantification.

Label-based quantification.

MS-based labelled quantification.

MS/MS-based quantification

Variance stabilizing transformations

Dynamic range.

Inferring relative quantity from peptide identification scores

Absolute quantification methods

Bibliographic notes

Peptides to Proteins

Peptides and proteins.

Protein identification using peptide masses: an example revisited

Extension to MS/MS derived peptide sequences instead of masses.

Minimal and maximal explanatory sets

Minimal and maximal sets in peptide-centric proteomics

Determining maximal explanatory sets.

Determining minimal explanatory sets.

Bibliographic notes.

Top-Down Proteomics

Separation of intact proteins.

Ionization of intact proteins.

Resolution and accuracy requirements for charge state determination and mass calculation.

Fragmentation of intact proteins

Charges of the fragments.

Protein identification.

Protein characterization - detecting modifications.

Problems with top-down approach

Exercises

Bibliographic notes

Standards

Standard creation.

Types of standards.

Standards from a proteomics perspective.

Creation of test samples

Data standards in proteomics

Requirements for data standards.

Problems with data standards

The Proteomics Standards Initiative (PSI).

Minimal reporting requirements

Mass spectrometry standards.

Modification standards.

Identification standards.

Bibliographic notes.

Bibliography

Index

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