IBiS 403. The Human Proteome: Defining Variations and Modifications

of Protein Molecules

Lecture 1Tuesday Jan. 6, 2015 1

INTERACTION PARTNERSSEQUENCE VARIANTSCOVALENT MODIFICATIONS

CANONICAL SEQUENCE

Some Logistics

Syllabus overview

• Distribution of assignments

• Reading expectations

• Course reserves

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NU New CMS: Canvas

(https://canvas.northwestern.edu/)

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https://canvas.northwestern.edu/

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Goal of Proteomics Course

To teach:

1. the value of proteomics

2. how such measurements are made

3. what questions can be answered

4. and current developments in the field of proteomics.

5. How proteomics could be used in (your) research

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Course Sections (Modules)

A. Mass Spectrometry

• Fundamentals and data collection

B. Bottom-Up Proteomics

• Data analysis and verification

C. Quantitative Proteomics

• Relative and absolute means

D. Top-Down Proteomics

E. Protein Variation

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Assumed Knowledge

Basic Eukaryotic Cell Biology• Central Dogma• Cell cycle• Signaling pathways

Basic Molecular Biology and Biochemistry• Molecular manipulations of DNA: PCR, cloning, transfection

etc.• Northern, Southern and Western blots

Basic Protein Chemistry and processing in vivo (a Must !)

mRNA processing, tRNA

ORFs, exons, introns, polymorphisms

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What is the proteome?

The complete set of unique protein molecules within a cell.

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From One Gene, Many Proteoforms

DNA

SNPs; translocations; gene families

mRNA proteins

~20,300 human genes

>100,000 mRNA >1,000,000 distinct proteoforms

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Splice variants Covalent modifications; proteolytic processing

Genome size of eukaryotes is

highly variable

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Genome size and gene number(C-value Paradox/Enigma)

The human genome has

minimal context

dependence; the human

proteome is the opposite

The genome is complicated

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The genome is complicated

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WHAT WE (MAINLY) CARE ABOUT IN THIS

COURSE

Sequence Variability

The Era of Functional Genomics

Human Genome Sequence

Drugs & Diagnostics

Expression Variability

Phenotypic variation

Mapping of Human Disease Loci

(Chromosomes 9-12)

http://www.ncbi.nlm.nih.gov/cgi-bin/SCIENCE96/chr?1

Nuclear membrane

Chromatin

fiber

}Trans-acting

factors

H1

H1

Nuclear matrix

Nuclear

pore

Interphase

nucleus

Nucleosomes

DNA

Chromatin fiber

(30 nm dia.)

By weight, the human genome is half DNA and half protein

Packaging of DNA into Chromatin

From Lodish et al. 2000.

Mosammaparast & Shi, 2010

Histone Modifications: Position- and State-specific

Eukaryotic gene structure (simplified)

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Alternative splice forms yield different

protein molecules

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Origins of Complexity in the Human Proteome

End processing

XPi

Polymorphism/`Mutations

CN

UnknownModifications

EnzymaticModifications

Variable Splicing

Ac Me

Ac

Key Concept: sources of protein variability result in a large, but finite number of protein forms (~106 / cell type), resulting in a measurement challenge.

What questions can proteomics answer?

What proteins are expressed?

How are these proteins modified?• Any covalent modifications?

• Alternative splice forms?

Where are these proteins located?• Which cell types?

• Which organelles?

When are these proteins expressed?• At what points in the cell cycle?

• In the presence of phamaceuticals?

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Mass Spectrometry is the workhorse for

untargeted proteomics

• High-throughput

• Confident identification of peptides/proteins

• Can achieve complete protein characterization

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