Proteomics 1

Introduction to Proteomics

  • Presenter Information

    • Doug Ward

    • Researcher in the Department of Cancer and Genomic Sciences, Birmingham

  • Session Objectives

    • Understand what proteomics is.

    • Describe the principles of proteomics workflow, including gel-based and shotgun proteomic techniques.

    • Explain the two main types of ionization techniques in mass spectrometry: electrospray ionization and MALDI (Matrix-Assisted Laser Desorption Ionization).

    • Identify peptides through mass spectrometry to infer protein identity.

Overview of Proteomics

  • Definition of Proteomics

    • Coined in 1994, proteomics is the study of the entire set of proteins (proteome) expressed in a biological model system, such as genes, tissues, or cell lines.

    • Distinction from DNA (genomic blueprint) is that proteins are dynamic, changing in response to different conditions and cellular environments.

  • Components of Proteomics

    • Proteome: A combination of “prot” (proteins) and “ome” (genome).

    • Modern Proteomics: Primarily based on mass spectrometry, differing from techniques that analyze single proteins, such as immunohistochemistry and western blots.

The Importance of Proteins

  • Functional Role of Proteins

    • Proteins are essential functional molecules: enzymes, receptors, structural proteins (e.g., cytoskeleton).

    • Their activity influences the metabolome and overall phenotype of cells, providing vital information that sequencing the genome and transcriptome cannot reveal.

  • Applications of Proteomics

    • Protein Interaction Analysis: Example procedure involving the antibody-mediated pull down technique.

    • Identifying proteins that bind to a target protein (e.g., protein B).

    • Protein Expression Comparison: Analyzing protein expression under different conditions (e.g., normoxia vs. hypoxia) either qualitatively or quantitatively.

Specific Applications of Proteomic Techniques

  • Analyzing Isoforms and Splice Variants

    • mRNA can generate multiple protein isoforms through splicing, necessitating direct examination of proteins to understand alternative splicing effects.

  • Post-Translational Modifications (PTMs)

    • Importance of PTMs such as phosphorylation, which influences protein function and signaling pathways.

    • Over 200 types of PTMs described, many identifiable using mass spectrometry.

  • Subcellular Localization

    • Proteomics allows for the determination of specific cellular locations (e.g., cytoplasm, organelles) where proteins function.

Identification of Proteins by Mass Spectrometry

  • Proteins as Linear Polymers

    • Composed of 20 amino acids, with varying molecular weights contributing to protein size (measured in kilodaltons).

  • Peptide Analysis

    • Proteins are typically broken into smaller peptides for easier identification.

    • Fragmentation of peptides yields sequence information crucial for identification (termed ‘bottom-up proteomics’).

Example of Mass Spectrometry in Practice

  • Trypsin Digestion

    • Most commonly used for mass spec is Trypsin. Digest proteins with trypsin, which specifically cleaves after every lysine (K) and arginine (R) residues.

    • Resulting peptides typically range from 7 to 25 amino acids, with a positive charge optimal for mass spectrometry.

  • Mass Spectrometer Process

    • Measurement of peptide mass results in a peptide mass fingerprint which can be put into a search engine for protein identification.

Workflow for Gel-Based Mass Spectrometry Digestion

  • Sample Preparation Steps

    • Run Gel electrophoresis and (SDS-PAGE) to separate proteins, then cut-out the protein band that your interested in for MS analysis.

    • Gel is dehydrated overnight and shrinks and trypsin is added

    • Disulfide bonds in protein are reduced using DTT

    • Followed by alkylation of cysteines to maintain the denatured state using iodacetamide.

    • Digestion with Trypsin

Alternative Sample Preparation Methods

  • In-Solution Digestion

    • With cases where the protein resists digestion due to tight folding:

    • The protein is denatured using 6-8M urea

    • Then digested with Trypsin and Lys-C mix for 3-4hrs. Lys-C digests proteins into large fragments. Trypsin doesn’t work here because of urea.

    • Dilute protein mix and Trypsin reactivates since urea is diluted

    • Then clean up of urea.

  • Filter-Aided Sample Preparation (FASP)

    • Utilizes a spin filter to separate proteins based on molecular weight, followed by digestion and collection of peptides through centrifugation.

Mass Spectrometry of Peptides.

  • Mass Spectrometry Components

    • Consists of an ion source, mass analyzer, and detector, connected to a computer for data analysis.

    • Measures mass-to-charge ratio of ions; requires conversion of peptides into ions.

  • Ionization Techniques

    • Two primary soft ionization techniques: Electrospray Ionization (ESI) and Matrix Assisted Laser Desorption Ionisation (MALDI)

MALDI Process

  • MALDI Mechanism

    • Small volume of sample is left on the MALDI target plate and allowed to dry.

    • ‘Matrix’- either CHCA for peptides or SPA for proteins is mixed in with sample and left to dry.

    • Matrix facilitates desorption and ionisation.

    • Plate is loaded into machine attached to time of flight mass spec.

  • Time-of-Flight MS

    • Positively charged ions are accelerated and analysed based on their time of flight through a vacuum; smaller ions travel faster to the detector. Measures the time it takes to hit detector.

  • Analysis of results: After MALDI-TOF, a list of peptide peaks will be made for each time a ion was detected. This list can be entered into a website/program called MASCOT which searches against a database of known sequences to get a match to identify our protein. MASCOT gives you a score to say how good your match is to the data base.

Electrospray Ionization (ESI)

  • Electrospray Principle - best for complex samples

    • After Liquid chromatography to get interested protein band

    • Liquid peptide sample is pushed through tiny needle, a high voltage is applied- creates a fine mist spray of charged droplets.

    • The droplets shrink as solvent evaporates, eventually releases charged peptide ions

    • Then go into TOF mass spec.

Tandem Mass Spectrometry (MSMS)

  • Process Overview

    • Isolates the peptides first via MS, then fragments for further identification using collision-induced dissociation (CID) to break peptide bonds

    • Then MS is done for the fragments, yielding Y ions (containing C-terminus) and B ions (containing N-terminus) used for sequencing.

Mass Spectrometers and Their Types

  • Common Mass Spectrometers

    • Types include Time-of-Flight (ToF), Quadrupole, Ion Trap, and Orbitrap.

  • Orbitrap: Ions are trapped in an orbit around a spindle shaped electrode and Fourier transform mathematics is used to convert image into mass/charge ratio. Main adv is most accurate and best MS, super accurate.

  • Quadrupole: Ions are mobilised by oscillating electric fields such that only those with a certain range of mass/charge ratio will pass through at one time. Main adv is super sensitive.

  • Ion trap: Ions are trapped within an electric field and allowed through the system in packets. And scans out to a detector. Main advantage is very fast and sensitive

  • TOF main adv is super simple and accurate.

Analysing Complex Mixtures- LC-MS/MS

  • Preliminary Steps- Separation of proteins

    • Need to simplify complex mixtures (e.g., blood plasma) via techniques such as two-dimensional gel electrophoresis and SDS-PAGE. Cut out gel ands and digest by in-gel digestion process. Identify by MALDI or LCMS

  • Shotgun Proteomics Approach

    • Peptide centric.

    • Complex sample is digested using trypsin

    • Separate peptides into multiple fractions

    • LCMS/MS analysis of fractions

    • Database search

    • ADV: more proteins detected than in electrophoresis, fast, sensitive, able to do comparisons.

    • DISADV: some structural info lost, only high specifications MS will give large numbers of identifications

Database Searching and Data Analysis

  • Search Engines and Databases

    • Use of tools like Mascot or Sequest to match experimental data against database sequences for protein identification based on mass and fragmentation patterns

  • Significance of Multiple Peptide Identifications

    • Greater confidence in identifications with multiple peptides suggesting presence; one-hit wonders should be treated with caution.

Conclusion

  • Proteomics reveals information beyond DNA and RNA sequencing capabilities, including protein interactions, isoforms, localization, and post-translational modifications.

    • Understanding mass spectrometry and sample preparation is crucial for performing proteomics research effectively.