The Syllabus

Safety First !!! Students will not be allowed into the lab without proper attire. Proper attire is designed for your protection and to avoid contaminations during experiments. In this class you must wear closed shoes, long pants, a lab coat and powder-free gloves . The latter will be provided in class.

Proteins are major structural and functional components of cells: Structural proteins (ex: Actin, Tubulin) Receptors (ex: Insulin, Adrenergic) Enzymes (ex: DNA polymerase, Hexokinase)

Protein Structure:

Many factors affect protein function in a given cell: Protein localization (ex: different organelles, intra or extracellular) Protein folding (ex: PTM, binding partners) Protein concentration (ex: Expression, half-life)

The Proteome: The proteome consists of the complete set of proteins found in a given cell. Different cells from a same organism will exhibit different proteomes (ex: Neurons vs Liver cells)

Proteome and Cell Differentiation: http://www.systembio.com/stem-cell-research/differentiation-reporters/overview

Genome vs Proteome: The genome encodes for the mRNAs that will give rise to the proteome The genome by itself does not provide enough information for a scientist to predict levels of protein expression, its binding partners, its half-life or subcellular location.

The Central Dogma of Biology: http://en.wikipedia.org/wiki/Central_dogma_of_molecular_biology

Genome vs Proteome: The genome is composed of four nucleotides. The proteome is composed of 20 aminoacids, plus post-translational modifications There are 256 (44) possible four nucleotide-long sequences, and 160,000 (204) possible four amino acid-long sequences (not counting PTMs!)

Proteomics: The aim of proteomics is to accurately identify and quantify proteins derived from tissues, whole cells, or even from subcellular localizations. Proteomics as we know it relies heavily on mass spectrometry for both qualitative and quantitative protein analysis.

General Approach for Proteomics Research: Sample Fractionation and preparation Sample Analysis Data Interpretation

Offline vs Online protein fractionation methods: Offline methods are used to fractionate samples prior to mass spectrometry analysis (Usually 1D or 2D SDS PAGE and/or Chromatography) Online methods are used simultaneously to mass spectrometry analysis (Chromatography)

Bottom Up vs Top Down Proteomics:

Protein Digestion: Usually with Trypsin (C-terminal of Arg, Lys – Endopeptidase only) Other enzymes include Chymotrypsin, Lysyl, Arg-C, Glu-C) Chemical digestion with CNBr

Protein Digestion with Trypsin: Very important for mass spectrometry!

Tryptic Peptides: Always have a terminal Arg or Lys with a fixed positive charge Peptides tend to be 20-40 aminoacids-long Products are easy to predict using simple bioinformatics

Mass Spectrometry: Can be thought of as being a “molecular scale”, which provides very precise readings of weight and quantity of molecules. Only works if molecules have at least one charge (either positive or negative) and are in the gas phase www.mayomedicallaboratories.com

The layout of a mass spectrometer: Source (MALDI, ESI,) Analyzer (TOF, Quadrupole, LTQ, Orbitrap, etc) Detector

The layout of a mass spectrometer: The source is designed to ionize samples and induce them to convert to the gas phase. The analyzer is designed to identify the molecule’s mass (in this case, m/z), and to fragment selected ions. The detector will quantify the molecules that reach it.

The ion source: Ion sources either perform “soft” or “hard” ionization Proteomics relies on sources which perform “soft ionization”, namely MALDI or ESI

Matrix-assisted LASER desorption/ionization (MALDI): http://prescottbiochem09.wikispaces.com

Electrospray Ionization: http://www.waters.com/waters

The Analyzer: There are many kinds of analyzers, with popular ones being: TOF, Quadrupole, LTQ and Orbitraps All of them need to be very accurate and precise For now we will focus on TOF instruments

Time-of-flight (TOF) analyzer:

The output of a Mass Spectrometer is called a “spectrum”: http://www.chemistry.ccsu.edu

The Spectrum: It provides you with the mass/charge ratio (m/z) of different molecules, of fragments of molecules The charge is given by the addition of a H+ to the molecule, adding 1 Da to its molecular weight (1000 + 1)/1 = 1001 m/z (1000 + 2)/2 = 501 m/z

Each amino acid has its own mass:

Different Algorithms will identify peptides based on their mass: Protein sequences can be deduced from genomic information. Proteins can be “virtually digested” with trypsin Proteomic information is also available on databases Each peptide can be identified trough the comparison of its mass to the theoretical ones

MS2 and MSn: If required, more structural information can be obtained through MSn analysis. In this case, chosen peptides will be fragmented in a collision cell within the analyzer. This will provide more information about amino acid residue composition and order within a peptide

We will discuss more about this and other subjects in the course of this semester.