Part II First 47 Flashcards
What are examples of what proteins are used for and give examples of
Catalysts (enzymes)
Transporters (of O2, membrane to take things in and out of cell)
Receptors (insulin, allergy receptors)
Structure builders (actin and myosin for muscles
Defense (antibodies for the immune system defense)
What is our definition of what proteins are
The machines and structural components of cells
How can some proteins be extracellular
They have sequences that tell them to go out of the cell (signal sequence)
What are some proteins that also have RNA in them
Ribosomes
Spliceosome
Why would we choose to purify proteins
Proteins control all biological function
Key in diseases so they’re very important to the pharmaceutical/biotechnology industry
In addition to making small molecule drugs, what do pharmaceutical companies now make
Give an example
Protein products
Ex. Monoclonal antibodies and vaccines
What are monoclonal antibodies (mABs)
A drug made of protein that targets other proteins
Since clinics mainly use monoclonal antibodies in drugs now, what does it helps with
Cancers, inflammation, genetic disorders, infectious agents
If drug has mab at the end it’s a
Monoclonal antibody
What is an antibody drug conjugate
What does it do
A monoclonal antibody with the drug covalently attached to it
This lets it target receptor proteins on the surface of the cell, In this way it can easily be taken in by the cell
Good for targeting cancer cells because it’s SPECIFIC and can get into those cells to destroy them
Majority of all approved drugs/antibodies are
What does this show
Monoclonal antibodies
This shows how powerful they are in treatments
What is the difference between genes and proteins
What is sequencing
Genes are mainly information storage units
Proteins carry out most cellular activities
Sequencing provides a parts list of that protein
What is the difference between genes and proteins
Genes are mainly information storage units
Proteins carry out most cellular activities
What is genome/RNA sequencing
Sequencing provides a parts list for protiens and can quantitatively tell us the levels of certain proteins in cells
It is quantitative, ex. Can tell that in certain cells with a certain condition, there were a certain number of transcripts for a particular protein
What is the definition of a proteome
The entire inventory of proteins that are produced by an organism
What does a cell proteomes variation depend on
CELL TYPE
Differentiation
developmental stage
Stresses
Cell cycle
Light vs dark
Nutrition status
What is the size of a Virus genome
Does prokaryotes have larger or smaller genome than eukaryotes
Small
Smaller
Virus>prokatore>eukaryotes
What special about c . Elegans and homosapein genome
Even though c elegans is still small, it has almost as many genes as a human
Both are eukaryotes
How many protein coding genes in humans
22000
How can genes give rise to several different proteins
Unconventional translation: Alternative splicing and alternative start sites in the main mRNA
Non canonical ORFs (open reading frames)
How can genes give rise to several different proteins
Unconventional translation: Alternative splicing and alternative start sites in the main mRNA
Non canonical ORFs (open reading frames)
How can the properties (function) of each protein be altered
By covalent modification
Since protiens are present in a wide changing range of amount and time they’re expressed across different cells
What makes a liver cell different than a muscle cell
They have the same genome but
The proteins each cell expresses is different which causes different metabolism in the cells
Muscle cells have more actin and myosin
What are proteoforms
What causes proteoforms
Different forms of proteins made from the genome due to variations in sequence
Gene isoform
Splice isoform (cutting out exons to make diff protien)
Non coventional translation (the mRNA has a different start site that is not AUG and translation starts from there)
Unrecognized ORF in mRNA
ORF in lncRNA (long non coding rna)
What are examples of PTMS
Phosphorylation, methylation, acetylation
On histones for example
What type of PTM does sars Covid have
Glycosylation to dock to other cells
How can we see what was being translated inside a cell
Explain what they found when doing this
We isolate the ribosomes during translation
This lets them identify unrecognized ORF and start sites
Found the sometimes the ribosomes would translated the unregconized ORF instead of the main ORF (forming micropeptides)
Found that the main function of this micropeptide (made from uORF) was linked to the function of the protein made from the main ORF
What is special about lncRNA (long non coding RNA)
What did the researcher find
They are conserved dna sequences that turn into rna but are non coding (we thought)
The researchers found that they actually are coding and form micropeptides
What makes micropeptides
Unrecognized open reading frame
LncRNA
Are all proteins expressed at the same level in different cells/tissues
No
What does the iceberg plot on slide 17 tell us
Only a few specific proteins are expressed at very high levels
As you get more and more different proteins, the number of copies of those different proteins gets lower and lower
Do proteins work alone
What do they do then
No
They specifically bind other proteins to form protein complexes
What’s the take away message from the proteins never do it alone article
Protein interactions are the rule , not the exception
The inside of the cell is
What does this mean for when we bust cells open
Very crowded
There is a massive dilution effect on the things in the cell
What are the reasons for purifying proteins
For research
For biotechnology / industrial scale purification
What are the reasons for purifying proteins for research
To purify something that’s responsible for a biological process
To characterize a protein by removing contamination factors
To check if it’s in a complex (most proteins are in complexes)
To identify and characterize domains and motifs that are responsible for the function of the protien
To do enzyme kinetics
To see the covalent modifications on it
To see the structure of it (by cloning then crystallizing it)
To raise antibodies agains the purified protein
Why would we want to remove contaminating factors in a protein
Because those factors could affect the activity of our protein of interest
Why would we want to remove contaminating factors in a protein
Because those factors could affect the activity of our protein of interest
What are the reasons for purifying proteins for biotechnology
For large scale production of therapeutic proteins
What is the Greek meaning of protein
Standing in front
What did Svedberg show
What did this help with
Showed that proteins can be separated by centrifugation
Making methods of protein separation like electrophoresis, affinity chromatography (AC) and ion exchange chromatography (IEX)
What chromatography system did the company pharmacia/cytvia make
FLPC (fast protein liquid chromatography)
What’s the difference in FPLC and HPLC
HPLC is high presssure
FPLC is low pressure
What is a heterologous protein
Taking a gene from one organism, putting it in another organism, and making it be expressed in that other organism
Resulting protein from that is a heterologous protein
Why is cell biology important to understand protein function
You need to know where the protien is in the cell or if it is even expressed in the cell before you can start to purify it
(This is why you need to think about the cell type you want to use)
Proteins are in specific organelles/locations to do specific roles (ex. TCA happens in mitochondria, so proteins for that would be in mitochondria)
Proteins are membrane bound (ex. The glogi membrane, plasma membrane, mitochondrial membrane
The cytosol is also considered a
Compartment in the cell
Why are there so many highly differentiatited cell in complex multicellular organism (like us and turkeys)
Because we have many different levels of protein expression in different cells
How many cell types in human
Over 200
What is special about cells in cultures
The can grown and reproduce for long periods of time
What are hela cells
The First Cultured tumor cells used for extended culturing (still being cultured)
They are cultured from a cancer patient Henrietta lacks
Why are cultured cells important for cell biologists
Because they can be used as a model for specific disease
And can be further studied
What is the difference between prokaryotes and eukaryotes
But what is the exception
Prokaryotes (bacteria) have rigid cell walls to protect the cell
But some eukaryotes also have cell walls
What is the difference between prokaryotes and eukaryotes
But what is the exception
Prokaryotes (bacteria) have rigid cell walls to protect the cell
Eukaryotic cells are more structurally and functionally complex than prokaryotes
But some eukaryotes also have cell walls
What eukaryotes have cell walls
Fungi, yeast, mold, mushroom, eukaryotic algae
What’s included in prokaryotes
What did eukaryotes evolve from
Bacteria and archaea
Archebacteria
What is similar in prokaryotes and eukaryotes
Because of the common ancestry they share
Identical Genetic language
Common Metabolic pathways and structural features
What similar in plant cells and prokaryotes
They both have plasma membranes that serve as a selectively permeable barrier
Both have cell walls
What bacteria has no cell walls
Microplasms
What are glycogen granules
In an animal cell, there are glycogen particles/granules
This is actually a subcomparent of the cell
Enzymes make and accumulate the glycogen in this compartment
What is the new organelles that is found in drosophila (flies)
A phosphate storing organelle
What is the nucleus in eukaryotic cells surrounded by
The membranous structure called the nuclear envelope
What it’s important about the cytoplasm of the cell
Forms a system of interconnecting channels that function to transport substances from one part of the cell to another
What is the nucleolus
The Timbit
Inside the nucleus
What is the consequences of busting cells open
A massive dilution effect (because previously crowded)
So now the activity of the enzyme dies and proteins start to unfold
What is special about the cellular localization of proteins
Where the protien is it’s important for its function (ex. Proteins in the nucleus are not in the cytosol)
However some proteins can undergo nuclear export and import (though those signal sequences that target them to specific parts of the cell)
When did mass spec begin
What is it
In 1912
Can find the mass of a molecule/molecule fragment to identify the molecule
What is proteomics
Exploring and analyzing fragments of proteins
What proteins are abundant in the cells
Ribosomes and histones
What is Maldi - TOF
How does it work
Matrix assisted laser desorption ionization time of flight
You have an unknown protein, treat it with trypsin (cleaves lys or arg at the carboxy term)
This makes them carry a postive charge
Send this sample down a flight tube where every peptide is separated by their mass to charge ratio (m/z)
Can say that based on the mass, we can say that the peptide can only have a specific comp of amino acids (to get that exact mass)
All of these different combo of amino acids in these peptides trace back to make the whole protien
What is maldi tof being replaced by
LC MS/MS
This is the actual sequencing of peptides by mass spec
What is protein sequencing by edman degredation
What type of amino acids elute later in HPLC
You have the full length protein and starts to sequence it starting from the n terminus
But this only sequences the n terminus and not the rest of the protein
So you digest the protein with trypsin
You react the solution with phenylisothiocyanate in high ph, this makes it attach to the n term of the peptide
Shifting it to low ph then releases a product which is a peptide shortened by one single n term
and makes a modified n term amino acid which you can do HPLC on to identify
Can keep doing this to get the entire protein sequence
More hydrophobic amino acids elute later in the HPLC
How do you prepared the sample for LC -MS/MS
You get your protein sample by cutting them out of a gel band (many protiens in the band)
Do trypsin digest and you get peptides from many different proteins
Put a charge on each peptide in the mixture through electro spray ionization
How does LC-MS/MS work
That charged peptide solution is sent down the mass analyzer which is the 1st flight tube in the machine
From this you get a MS spectrum and you take a single peptide from this
That single peotide is sent to the collision chamber where it’s fragmented (via neutral argon gas)
Then this fragemnted peptide is sent down a second flight tube where a MS/MS spectrum of it it made
How do you analyze a LC- MS/MS spectrum
During the collision, the ions are being taken off the n terminus of the selected peptide (y series ions)
The aa are being taken off the c terminus (B series ion)
So B and Y series ions are seen in the MS/MS spectrum
The difference between each of these series (ex. Y2 vs Y1) is the mass of one amino acid
So this specific mass difference (loss) can tell you what amino acid was in that part of the sequence
In LC MS/MS what special thing happens to the B ion
During collision is can sometimes lose a carbonyl group to form an a type ion
In LC MS/MS what special thing happens to the B ion
During collision is can sometimes lose a carbonyl group to form an a type ion
What major peptides do we see in LC MS/MS
B and Y
Formed when the amide/peptide bond is broken
What is de novo peptide sequencing
The b series ions from LC MS/MS are analyzed
This is where the amino acid is cleaved from the c terminus
The exact mass difference in the next b series vs the last corresponds to which amino acid is missing (ie the next amino acid in the sequence)
In LC MSMS what if the mass loss didn’t match the mass of any aminos acid
How is this useful
This indicates that the amino acid have been covelently modified
This is how proteomics is useful cause you can identify PTM
What’s the most used electrophoresis method
What does it do
SDS page
The sds (sodium dodecyl sulfate) is a anionic detergent that denatures and reduces the proteins (gives them all negative charge)
This makes it so that Protiens are separated by mass only and not charge (bigger slower)
What is nondenaturing page
Why is it useful
Also called native page
Separates proteins based on net charge, mass, and shape of their native structure
Higher negative charge, faster protein migrates due to more negative. Due to mass to charge ratios (smaller mass, more negative, faster)
No denarurant used , meaning Protiens keep their enzymatic activity
This is why this can be used for protein purification
What is 2D PAGE
Separates proteins by their isoelectric point in one dimension
And by mass in the other
So in a ph gradient, the protiens that reach their isoelectric point stop migrating at that ph
Then that ph gradient strip would be put on a SDS page to separate by mass
In SDS page, what is added to removes questernatry/tertiarry structure
Reducing agent (DTT/2-ME)
This cleaves the disulphides bond
This makes it so that it’s for sure only mass and not shape that is affecting how they move
What are the samples in SDS page treated with to see
Tracking dye to see the migration (electrophoretic movement) of the sample
What in a SDS page sample buffer
Tris HCL and ph 6.8 (buffer to keep at neutral ph)
Glycerol (so that the sample sink into the well and doesn’t spill into other wells)
SDS (deterring that makes proteins uniformly negative charged)
2ME (reduces disulfide bonds)
Bromophenol blue (negative charged, low MW dye)
What in a SDS page sample buffer
Tris HCL and ph 6.8 (buffer to keep at neutral ph)
Glycerol (so that the sample sink into the well and doesn’t spill into other wells)
SDS (deterring that makes proteins uniformly negative charged)
2ME (reduces disulfide bonds)
Bromophenol blue (negative charged, low MW dye)
What is special about bromophenol blue
It’s used as a PH indicator as well
sample turns green when you add the dye, this means the sample is too acidic and ph need to be change
In native page, if you had a band at 100kda and one at 20 for one sample what does this mean
100/20 = 5
This means that the protein had five identical subunits each with 20kda mass
SDS page show what type of protein structure
Monomeric
All purification of proteins involves
Chromatography
And separating the protein of interest based on properties of other proteins in the sample
What property do you use to purify a protein via affinity chromatography
What type of specific can it be
Specific ligand recognition
Bio specific (attaching to atp)
Non bio specific (attaching to dyes)
What property do you use to purify a protein via IMAC
The metal ion binding of the protein (when a tag is on it ex his tag)
What property do you use to purify a protein via IEX
SEC/gel filtration
HIC/reverse phase chromatography (RP)
Charge
Size (using the entire fractionation range of the protein solution which you can use to get your target protein)
Hydrophobicity
What are the main methods we exploit to purify proteins
Charge, size, hydrophobicity
What is the general workflow of a purification
Extraction or homogenize the cells/tissue
Centrifuge (get rid of cell debris)
PEG (polyethylene glycol) or ammonium sulfate PPT. (To precipitate our protein of interest)
Resuspend pellet
Capture through 1st chromatography
Then do a second and third chromatography
Where do you get the source tissue/cells for the first step of the general workflow of purification
Bacterial cells
Tissue
Human cells
Insect cells
Or other niche organisms
How do you find the protein after doing the chromatography step
Assays of each fraction for activity, A280
Do ads page or western blot is you know the proteins mass
What are expression vectors
But before making an expression vector, the protein has to be
Forcing the bacteria to make our protein of interest
Purified from endogenous sources
What are the endogenous sources that proteins can be purified from
Whole organisms (yeast/bacteria)
Tissue or organ (skeletal muscle/liver)
Subconpartment of a specific cell type/tissue (mitochondria, glycogen particle)
How do we modify proteins to make them easier to purify
Clone then tag them (6HIS, GST, MBP)
Then over express them in a new host
Then purify them with a tag
Tagged proteins are detected by western blot with a antibody to
The tag
What an example of how you can do an assay to see Hexokinase activity
Hexokinase turns glucose into G6P
Can couple the reaction with G6PDH
G6PDH turns G6P into 6-P-gluconate by trying NADP to NADPH
NadPh absorbs at 340nm so you can measure amount of Hexokinase activity by amount of NADH absorbing
