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Proteomics (Omics)

LECTURE 5: INTRODUCTION INTO METAPROTEOMICS

1. THEORY METAPROTEOMICS
Metaproteomics = microbial community proteomics

What would we like to know about microbial communities?

- What species are there? How many? (taxonomy) →

Difficult to know since there may be hundreds or
thousands of species.
- What are their substrates? What do they eat/consume?
(Glucose, fructose, CO2…)
- Which proteins (pathways) are expressed for feeding?
- What eats each specie?

CHALLENGES IN METAPROTEOMICS
1) Proteome complexity. From which species the protein comes? Remember that each yeast
species has like 6000 proteins, so with 100 species in a community, we would have 600.000
proteins...
2) Protein extraction. There are many different cell types, and the same extraction method does
not work for all of them.
3) Reference sequence database: many unsequenced species.
4) Functions: possibly unknown metabolic routes, many new enzymes.

Metaproteomics usually employs the conventional bottom-up shotgun proteomics approach: the
peptides obtained in the digestion of the community proteins are compared in databases to find the
protein sequences.

Where do we get the protein sequence reference database?

• Option 1: from metagenomics. We sequence massively the genomes of the microorganisms in

the sample.
o Disadvantages:
▪ Extract DNA from a certain part of the community.
▪ You amplify all the DNA, the one that is in very small amounts and reverse, so
you don't really know what the most abundant specie in the community is.
▪ High costs
▪ Time consuming

• Option 2: use generic reference sequence databases such as NCBI and UniProt.
o Advantage: no amplification, only the proteins that are sufficiently high abundant will
be detected. Realistic data from the community.
o Disadvantage: you will not find new species since you are comparing the peptides
sequences with known proteins.
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Proteomics (Omics)

2. APPLICATIONS
WHO IS THERE? WHO DOES THAT?

From an environmental sample, a metaproteomics (and maybe a metagenomics) experiment is

carried out to identify the proteins and, therefore, the species.

WHO IS ACTIVE? WHICH MICROBE EATS WHAT?

Protein stable isotope probing (SIP) allows to measure utilisation of substrates by individual
microbes. A stable isotope (i.e., C13) labelled substrate is used to feed the microbes, so the ones that
consume it will assimilate and acquire the label.

Ex: labelled glucose. It is consumed and the carbon finishes in the aminoacids, so it can be detected
later.

Ex: peptides mass spectrum which shows stable isotope incorporation over time. Each coloured peak
corresponds to a specific time: more time = more assimilation of the stable isotope.
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Proteomics (Omics)

Two key parameters in stable isotope probing: RIA and LR

RIA = Relative isotope abundance LR = Labelling ratio
How much 12C has been replaced by 13C? What is the fraction of labelled proteins?

The peak moves to the right each time we have

another 13C (m/z increases because mass
increases).
If you don't really see a big move to the right, it's
LR let us know how fast the microbe is in
because the microbe is using another feeding
terms of making new proteins, because
source apart from the one you labelled (there is a
this ratio expresses the assimilation of
2nd carbon source or multiple carbon sources).
labelled carbons.
The more a labelled carbon source is consumed,
the higher is RIA. But if the microbe uses other
carbon sources, it will assimilate the labelled carbon
source more slowly so its RIA will be 'smaller'.