Proteomics - created from Mind Map

Página 1

<ul><li>less than 1.5% of the human genome encodes proteins and most of the genome is transcribed to RNA </li></ul>

genotype<br><br><ul><li>Genome is the blueprint of

what could be made.</li></ul>

phenotype<br><br><ul><li>Phenotype is what is made.

– mRNA expression.

– Non-coding RNA.

– Proteins and proteoforms.

– Lipids.

– Metabolites.</li></ul>

different genes expressed in different cells<br><br><ul><li>We identified a total of 11,731 proteins, and on average 10,361 ± 120 proteins in each cell line. This very high proteome coverage enabled analysis of a broad range of processes and functions. Despite the distinct origins of the cell lines, our quantitative results showed surprisingly high similarity in terms of expressed proteins.</li><li>lobal similarity of the proteomes did not imply equal expression levels of individual proteins across the 11 cell lines, as we found significant differences in expression levels for an estimated two-third of them. The variability in cellular expression levels was similar for low and high abundance proteins, and even many of the most highly expressed proteins with household roles showed significant differences between cells. Metabolic pathways, which have high redundancy, exhibited variable expression, whereas basic cellular functions such as the basal transcription machinery varied much less.</li></ul>

genome --> transcriptome<br><br><ul><li>Whole genomes sequenced in

less than a wee</li><li>Multiple forms of mRNA from

one gene.

– Multiple forms of primary

protein sequence.

– Proteoform.

• Transcriptome: Abundance of

different mRNA’s in a cell type</li></ul>

more proteins than genes

fractionation<br><br><ul><li>uantity of a mixture (gas, solid, liquid, enzymes, suspension, or isotope) is divided during a phase transition, into a number of smaller quantities (fractions) in which the composition varies according to a gradient.a process which a certain </li><li>Fractionation is necessary to reduce sample complexity

and dynamic range of protein concentration</li><li>Use chemical properties to reduce sample complexity</li></ul>

same genome, different proteome

whats the proteome?<br><br><ul><li>The Proteome is ALL of the proteins expressed by a cell,

organism or tissue under a certain set of environmental

conditions.

• Genomics measures Genotype.

• Proteomics is a measure of Phenotype.

– Phenotype also defined by metabolites (metabolome), lipids

(lipidome).

• A cell is NOT just what proteins are expressed, but:

– When is the protein expressed and how much?

– How is it modified post-translation?

– What is the protein interacting with?</li></ul>

what is it?

Analysis of proteome<br><br><ul><li>How is the Proteome analysed?

• Proteomics uses well established methodologies to

fractionate and separate proteins from one another.

– Electrophoresis.

– Chromatography.

• Mass spectrometry (MS) is used to produce data

that reflects protein/peptide amino acid sequence.

• MS data is “matched” using algorithms to known or

predicted protein sequences.</li></ul>

technologies

Workflow

if unfractionated?

problems

peptide vs. protein

quantitating changes in proteome

examples

multiple forms of mRNA from one gene

transcriptome

post-translational cleavage

estimation of proteoforms

size

charge

hydrophobicity

different from genomics

what does proteomics study?<br><br><ul><li>Obtain a global view of cell or organism at the

protein level under a certain environmental

condition.

– Measure quantitative differences in amount of protein

expressed.

• Includes analysis of the modifications possible on

proteins, including cleavage, phosphorylation,

glycosylation, lipidation, etc.

• The challenge of proteomics is to quantify the

changes in proteins when the system they are

part of changes.</li></ul>

not just what proteins are expressed

challenges

why study it?<br><br><ul><li>• To determine phenotypic changes in a system.

• To ascertain the function and interactions that

proteins have in normal, diseased and infected

cells.

• Determine mechanisms of cellular function.

• How do protein interactions and complexes alter

in disease?

• Find targets for therapeutic intervention.

• Find “Biomarkers” for early diagnosis of disease.</li></ul>

PCR<br><br><ul><li>Genomics/Transcriptomics – can ‘amplify’ signal using PCR.

– Targeted amplification of low abundance mRNA.

• Proteins – no equivalent of PCR.

• Want to detect these, start with more.

• But more of these, overwhelming signal.

– Back where you started.

– And if you can’t get more? </li></ul>

electrophoresis

Chromatography

Mass Spec

smashing cells open<br><br><ul><li>Smash everything open.

• Divide the protein into manageable groups

• Cut it up to peptides.

• Measure peptide mass in the MS and smash

the peptide to measure the fragments.

• Search databases for matches.

• Interpret what it all means biologically.</li><li>A. Do you want to catalogue everything?

B. Do you want to find out what proteins are

interacting?

• Often you can’t have both.

– To analyse a protein, first you have to get to it and

second you have to solubilise it.</li></ul>

how to interpret data

2D gel

isoelectric focusing

output meaning

intact spectra

fragmented spectra

searching/matching MS data

fragmentation

heterogenity<br><br><ul><li>Heterogeneity of proteins means each workflow

is tailored to the particulars of the sample and

the subset of proteins needing analysis.</li></ul>

Solubilise<br><br><ul><li>proteins so they can be fractionated</li></ul>

Fractionate<br><br><ul><li>Fractionate proteins by their differing physical

properties.</li></ul>

Analyse<br><br><ul><li>Analysing unfractionated samples displays only the

highest abundance proteins.

– 2-300 proteins seen of 23,000+ proteins and 500,000+

proteoforms.

identify and quantify. </li></ul>

theoretical peptide mass=measured mass

if sequence not in database

peptide for difinition not detected

how many proteins expressed

ID

amount

measure

mass spec

2D-PAGE

comprehensive ms based proteome quantification of haploid versus diploid yeast

plasmodium falciparum proteome changes in reponse to treatment