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Bchem 411 > Part II First 47 > Flashcards

Part II First 47 Flashcards

1
Q

What are examples of what proteins are used for and give examples of

A

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)

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2
Q

What is our definition of what proteins are

A

The machines and structural components of cells

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3
Q

How can some proteins be extracellular

A

They have sequences that tell them to go out of the cell (signal sequence)

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4
Q

What are some proteins that also have RNA in them

A

Ribosomes

Spliceosome

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5
Q

Why would we choose to purify proteins

A

Proteins control all biological function

Key in diseases so they’re very important to the pharmaceutical/biotechnology industry

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6
Q

In addition to making small molecule drugs, what do pharmaceutical companies now make

Give an example

A

Protein products

Ex. Monoclonal antibodies and vaccines

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7
Q

What are monoclonal antibodies (mABs)

A

A drug made of protein that targets other proteins

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8
Q

Since clinics mainly use monoclonal antibodies in drugs now, what does it helps with

A

Cancers, inflammation, genetic disorders, infectious agents

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9
Q

If drug has mab at the end it’s a

A

Monoclonal antibody

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10
Q

What is an antibody drug conjugate

What does it do

A

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

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11
Q

Majority of all approved drugs/antibodies are

What does this show

A

Monoclonal antibodies

This shows how powerful they are in treatments

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12
Q

What is the difference between genes and proteins

What is sequencing

A

Genes are mainly information storage units

Proteins carry out most cellular activities

Sequencing provides a parts list of that protein

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13
Q

What is the difference between genes and proteins

A

Genes are mainly information storage units

Proteins carry out most cellular activities

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14
Q

What is genome/RNA sequencing

A

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

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15
Q

What is the definition of a proteome

A

The entire inventory of proteins that are produced by an organism

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16
Q

What does a cell proteomes variation depend on

A

CELL TYPE

Differentiation

developmental stage

Stresses

Cell cycle

Light vs dark

Nutrition status

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17
Q

What is the size of a Virus genome

Does prokaryotes have larger or smaller genome than eukaryotes

A

Small

Smaller

Virus>prokatore>eukaryotes

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18
Q

What special about c . Elegans and homosapein genome

A

Even though c elegans is still small, it has almost as many genes as a human

Both are eukaryotes

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19
Q

How many protein coding genes in humans

A

22000

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20
Q

How can genes give rise to several different proteins

A

Unconventional translation: Alternative splicing and alternative start sites in the main mRNA

Non canonical ORFs (open reading frames)

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21
Q

How can genes give rise to several different proteins

A

Unconventional translation: Alternative splicing and alternative start sites in the main mRNA

Non canonical ORFs (open reading frames)

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22
Q

How can the properties (function) of each protein be altered

A

By covalent modification

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23
Q

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

A

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

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24
Q

What are proteoforms

What causes proteoforms

A

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)

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25
Q

What are examples of PTMS

A

Phosphorylation, methylation, acetylation

On histones for example

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26
Q

What type of PTM does sars Covid have

A

Glycosylation to dock to other cells

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27
Q

How can we see what was being translated inside a cell

Explain what they found when doing this

A

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

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28
Q

What is special about lncRNA (long non coding RNA)

What did the researcher find

A

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

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29
Q

What makes micropeptides

A

Unrecognized open reading frame

LncRNA

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30
Q

Are all proteins expressed at the same level in different cells/tissues

A

No

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31
Q

What does the iceberg plot on slide 17 tell us

A

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

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32
Q

Do proteins work alone

What do they do then

A

No

They specifically bind other proteins to form protein complexes

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33
Q

What’s the take away message from the proteins never do it alone article

A

Protein interactions are the rule , not the exception

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34
Q

The inside of the cell is

What does this mean for when we bust cells open

A

Very crowded

There is a massive dilution effect on the things in the cell

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35
Q

What are the reasons for purifying proteins

A

For research

For biotechnology / industrial scale purification

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36
Q

What are the reasons for purifying proteins for research

A

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

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37
Q

Why would we want to remove contaminating factors in a protein

A

Because those factors could affect the activity of our protein of interest

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38
Q

Why would we want to remove contaminating factors in a protein

A

Because those factors could affect the activity of our protein of interest

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39
Q

What are the reasons for purifying proteins for biotechnology

A

For large scale production of therapeutic proteins

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40
Q

What is the Greek meaning of protein

A

Standing in front

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41
Q

What did Svedberg show

What did this help with

A

Showed that proteins can be separated by centrifugation

Making methods of protein separation like electrophoresis, affinity chromatography (AC) and ion exchange chromatography (IEX)

42
Q

What chromatography system did the company pharmacia/cytvia make

A

FLPC (fast protein liquid chromatography)

43
Q

What’s the difference in FPLC and HPLC

A

HPLC is high presssure

FPLC is low pressure

44
Q

What is a heterologous protein

A

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

45
Q

Why is cell biology important to understand protein function

A

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

46
Q

The cytosol is also considered a

A

Compartment in the cell

47
Q

Why are there so many highly differentiatited cell in complex multicellular organism (like us and turkeys)

A

Because we have many different levels of protein expression in different cells

48
Q

How many cell types in human

A

Over 200

49
Q

What is special about cells in cultures

A

The can grown and reproduce for long periods of time

50
Q

What are hela cells

A

The First Cultured tumor cells used for extended culturing (still being cultured)

They are cultured from a cancer patient Henrietta lacks

51
Q

Why are cultured cells important for cell biologists

A

Because they can be used as a model for specific disease

And can be further studied

52
Q

What is the difference between prokaryotes and eukaryotes

But what is the exception

A

Prokaryotes (bacteria) have rigid cell walls to protect the cell

But some eukaryotes also have cell walls

53
Q

What is the difference between prokaryotes and eukaryotes

But what is the exception

A

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

54
Q

What eukaryotes have cell walls

A

Fungi, yeast, mold, mushroom, eukaryotic algae

55
Q

What’s included in prokaryotes

What did eukaryotes evolve from

A

Bacteria and archaea

Archebacteria

56
Q

What is similar in prokaryotes and eukaryotes

A

Because of the common ancestry they share

Identical Genetic language

Common Metabolic pathways and structural features

57
Q

What similar in plant cells and prokaryotes

A

They both have plasma membranes that serve as a selectively permeable barrier

Both have cell walls

58
Q

What bacteria has no cell walls

A

Microplasms

59
Q

What are glycogen granules

A

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

60
Q

What is the new organelles that is found in drosophila (flies)

A

A phosphate storing organelle

61
Q

What is the nucleus in eukaryotic cells surrounded by

A

The membranous structure called the nuclear envelope

62
Q

What it’s important about the cytoplasm of the cell

A

Forms a system of interconnecting channels that function to transport substances from one part of the cell to another

63
Q

What is the nucleolus

A

The Timbit

Inside the nucleus

64
Q

What is the consequences of busting cells open

A

A massive dilution effect (because previously crowded)

So now the activity of the enzyme dies and proteins start to unfold

65
Q

What is special about the cellular localization of proteins

A

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)

66
Q

When did mass spec begin

What is it

A

In 1912

Can find the mass of a molecule/molecule fragment to identify the molecule

67
Q

What is proteomics

A

Exploring and analyzing fragments of proteins

68
Q

What proteins are abundant in the cells

A

Ribosomes and histones

69
Q

What is Maldi - TOF

How does it work

A

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

70
Q

What is maldi tof being replaced by

A

LC MS/MS

This is the actual sequencing of peptides by mass spec

71
Q

What is protein sequencing by edman degredation

What type of amino acids elute later in HPLC

A

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

72
Q

How do you prepared the sample for LC -MS/MS

A

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

73
Q

How does LC-MS/MS work

A

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

74
Q

How do you analyze a LC- MS/MS spectrum

A

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

75
Q

In LC MS/MS what special thing happens to the B ion

A

During collision is can sometimes lose a carbonyl group to form an a type ion

76
Q

In LC MS/MS what special thing happens to the B ion

A

During collision is can sometimes lose a carbonyl group to form an a type ion

77
Q

What major peptides do we see in LC MS/MS

A

B and Y

Formed when the amide/peptide bond is broken

78
Q

What is de novo peptide sequencing

A

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)

79
Q

In LC MSMS what if the mass loss didn’t match the mass of any aminos acid

How is this useful

A

This indicates that the amino acid have been covelently modified

This is how proteomics is useful cause you can identify PTM

80
Q

What’s the most used electrophoresis method

What does it do

A

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)

81
Q

What is nondenaturing page

Why is it useful

A

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

82
Q

What is 2D PAGE

A

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

83
Q

In SDS page, what is added to removes questernatry/tertiarry structure

A

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

84
Q

What are the samples in SDS page treated with to see

A

Tracking dye to see the migration (electrophoretic movement) of the sample

85
Q

What in a SDS page sample buffer

A

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)

86
Q

What in a SDS page sample buffer

A

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)

87
Q

What is special about bromophenol blue

A

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

88
Q

In native page, if you had a band at 100kda and one at 20 for one sample what does this mean

A

100/20 = 5

This means that the protein had five identical subunits each with 20kda mass

89
Q

SDS page show what type of protein structure

A

Monomeric

90
Q

All purification of proteins involves

A

Chromatography

And separating the protein of interest based on properties of other proteins in the sample

91
Q

What property do you use to purify a protein via affinity chromatography

What type of specific can it be

A

Specific ligand recognition

Bio specific (attaching to atp)

Non bio specific (attaching to dyes)

92
Q

What property do you use to purify a protein via IMAC

A

The metal ion binding of the protein (when a tag is on it ex his tag)

93
Q

What property do you use to purify a protein via IEX

SEC/gel filtration

HIC/reverse phase chromatography (RP)

A

Charge

Size (using the entire fractionation range of the protein solution which you can use to get your target protein)

Hydrophobicity

94
Q

What are the main methods we exploit to purify proteins

A

Charge, size, hydrophobicity

95
Q

What is the general workflow of a purification

A

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

96
Q

Where do you get the source tissue/cells for the first step of the general workflow of purification

A

Bacterial cells

Tissue

Human cells

Insect cells

Or other niche organisms

97
Q

How do you find the protein after doing the chromatography step

A

Assays of each fraction for activity, A280

Do ads page or western blot is you know the proteins mass

98
Q

What are expression vectors

But before making an expression vector, the protein has to be

A

Forcing the bacteria to make our protein of interest

Purified from endogenous sources

99
Q

What are the endogenous sources that proteins can be purified from

A

Whole organisms (yeast/bacteria)

Tissue or organ (skeletal muscle/liver)

Subconpartment of a specific cell type/tissue (mitochondria, glycogen particle)

100
Q

How do we modify proteins to make them easier to purify

A

Clone then tag them (6HIS, GST, MBP)

Then over express them in a new host

Then purify them with a tag

101
Q

Tagged proteins are detected by western blot with a antibody to

A

The tag

102
Q

What an example of how you can do an assay to see Hexokinase activity

A

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

Bchem 411 (9 decks)

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