proteomics Flashcards
1
Q
how many aa are there?
A
20
2
Q
which aa is named after silk
A
serine
3
Q
which aa is pro
A
proline- contraction of pyrrolidine
4
Q
what amino acid is names after roots of plants?
A
valine
5
Q
what aa is named after the colour white
A
leucine
6
Q
which aa is gln
A
glutamine
7
Q
which aa is named after a structurally similar aa
A
isoleucine
8
Q
which aa was first found in old cheese
A
tyrosine
9
Q
which aa is named after bladder?
A
tyrosine
10
Q
which aa is met
A
methionine
11
Q
what are the two aa ending in acid?
A
aspartic acid and glutamic acid
12
Q
which aa is named after juice?
A
asparagine
13
Q
which aa was hypothesised to be similar to another?
A
glutamine
14
Q
what are peptides held together by
A
peptide bonds
15
Q
how many aa are a helix
A
10
16
Q
how many aa in beta pleated sheet
A
6
17
Q
what is the size of phenylalanine hydroxylase
A
108kDA
18
Q
in 2 ul of plasma how much protein is there
A
1060 unique proteins
19
Q
how big is a growth hormone
A
22 kDa
20
Q
How big is ferritin
A
42 kDa
21
Q
how big is a single ferritin unit
A
480 kDa
22
Q
which direction does the b ions go from
A
left to right
23
Q
what direction does the y ion go from
A
right to left
24
Q
how are b and y ions made
A
electrospray ionisation
25
why do we need high reolsultion for mass spec
so that you can tell the difference as some will have the same mass but different chemical structures at low resolution cannot tell the difference
26
what is mass resolution
ability to resolve closely adjacent peaks
27
what is the advantage of madli matrix
can analyse high masses so big proteins can lookout changes in proteins in transit modifications as can look at the whole protein
28
what are the disadvantages of maldi
can only look at one protein at a time and so has limited throughput doesn't use MS2 so have to caluclate mass
29
how many common proteins are found in the blood?
289
30
how would you analyse complex mixtures
would use electrophoresis to break it down into fragments so easier to analyse.
31
how many proteins detected in the human proteome atlas
30k
32
how many peptides are detected in the human proteome atlas
290k
33
what are the methods for protein estimation
-bradord assay
-lowry method
-bicinchronic acid assay
-flourscent based assay
all rely on liaise
34
what is the sample prep for proteomic analysis
protein extraction, estimation and purification immunoprecipiatation, trypsin digestion and clean up
35
which mass anaylzer has the highest relation
orbi trap
36
what is mass resolution
the ability to resolved closely related adjunct mass peak. The bigger the number the higher the mass resolution
37
what is scan speed
how fast the mass analyser scan the entire range of mass spec
38
which modes would you use on mass spec if you were looking for specific peptides
MRN and PRM
39
what mass spec mode would you use for discovery work (identifying as many polypeptides as possible)
DDA and DIA
40
How does DDA work?
relys on chromatography to separate complex mixtures and elutes the most abundant- easier to use for discovery work
41
how does DIA work
splits actual space into blocks and fragments everything has higher fidelity
42
what do we need to do for mass spec
they look at protein mixtures so need to unfold protein and typsinise them
43
what are the advantages of madli
looks at intact proteins. Can look at big proteins as has a high mass range
44
what is the disadvtange of madli
can only look at one protein at a time as proteins have multiple charge states so if looked at multiple wouldn't know which protein it is coming from
doesn't have MS2 so can take days to analyse
45
how does MALDI work
laser burns through the matrix and into sample but make sure doesn't degrade samples. It ionises and the ions travel down TOF.
46
how many proteins have been identified in the human body
30k
47
what can you do to complex mixtures to make it more basic
can fractionate mixtures into crude molecular sizes by gel electrophoresis
48
what is the limitation of proteomics
can only identify 60-80% of proteins as they will fall below detection
49
how many peptide sequences are in the body?
290k
50
what lipase techniques are used for protein estimation
-lowry method
-bradford assay
-bicinchronic acid assay
-fluoroscent based assay
51
what do you want to know before testing a sample
a rough estimate of protein in the sample so taht you know how much topspin to include
52
how do you do a rough estimate of protein in a sample
calibration curve of the known conc of protein and then profile unknown profiles, dont want to quantify the protein where it isn't linear. correlation of coefficient should be 0.98
53
what is the ratio of tryspsin you want to use
20-50:1
54
what is the gel base fractoination method
make the gel and adjust the pore size on ratio of amide. want to separate into crude fraction. In each band there is one band there is one class of protein not one type
55
what are steps of shot gyn proteomic analysis
-this is done on peptides
extraction of protein
-subsequent determination of approximate protein- Bradford assay
-fractionantion and trypsin digestion
-clean up step and lyophilisation
-chemical analysis (DIA or DDA)
-database matching and peak table generations
-data analysis and interpretation
56
what does shot gun proteomics tell you
won't know which fragment belongs to which protein. instead can see peptide structures being eluted so know method is working
57
what is the optimum temp for trypsin digestion
37 degrees
58
how does trypsiin work
cleaves the C term of the K R unless next to proline
59
what do you have to do to sample before trypsin
denture the protein in 6-8 M of urea
60
what do you do with trypsin digestion
suspend in ammonium bicarbonate
digest with trypsin and lys-c
trysin has been inactivated by urea
lys-c digests protein into large fragments
After dilution and longer incubation trypsin reactivates and completes digestion
61
what is reposnivle for seperation in a highly reproducible manner
LC front end
62
what is included to allow mass measurement and determination
electric/magnetic field, vacuum, super shaped electrodes and high speed switching micro electronics
63
how is LC chromotographic done
analytical LC proteomic column and solvent gradient profile
64
what are the results from a mass analyser
quantify of peptide abundances on the form of m/z ratio and record the spectral repines as function of total ion current against retention time
65
explain the electro spray ionisation of the analyte
supply high voltages of 3-4 kv at tip of spray needle. This creates a coulombic explosion creating analyte ions. want mobile phase to evaporate straight way as most abundant
66
what is in silco
idealised version of what we want to see. but doesn't ge the real csontrastof the real world like ion suprreaion and the complexity of ionisation. But if your results match it then can be used for protein identification
67
what are the advantages of top down proteomics
- intact protein analysis
-de novo sequencing
-indenitifation of protein isoforms
-lwers chance of false positive
-100% protein sequence coverage is possible
-characterisation of proteolytic processing events and post translational modification
68
what are the disadvantages of top down proteomics
-lower sensitivity and throughput
-high sample purity required
-insoluble proteins and big proteins not amenable
-expenisve
-complex data and information
-sample prep
-operator skill
69
what is the advantages of bottom up proteomics
-more complex mixtures can be analysed
-higher throughput
-greaterlevel of chemical/biological fucbntion can be obtained
-global coverage and characterisation of biological system
-ms2 level information, ID and database matching
-greater level of biological information and interpretation
-greater analytical flexibility
70
what is are examples of bottom up proteomics
shot gun
71
what is top down proteomics
high resolution mass spec - intact protein analysis
72
what are the disadvantages of bottom up
-chemical identification
-high rate of false positive
-protwins without tryptic peptides within the right masss window are missed
-pmt and isoform information often less without correct analytical stratergy
sample prep
-complex system and maintains, calibrations and system performance assessment
73
What is the definition of proteome
Profiling an entire set of proteins within a system/cell at a given time and defined conditions
74
What is a limitation of proteomics
60-80% detection
75
How do you increase protein coverage after gel electrophoresis
By using LC/MS methodology
76
what do the mass analyses do
they detect, measure and quantify the peptides abundances in the form of m/z ratio and record the spectral response as function of TIC against retention time
77
what is the disadvantages of top down/maldi
low sensistivity
high sample pruirty needed
operator skill
expensive instruments.
78
what are the advantages of bottom up proteomics
- can analysis more complex mixtures
higher throughput
greater level of chemical and bioodiucal function can be obtained
MS2 information
greater anyalytical flexibity
79
what are the disadvantages of bottom up
high rate of false positive
sample prep
complex system
proteins withoit tryptic proteins within the right mass window missed
79
what are the disadvantages of bottom up
high rate of false positive
sample prep
complex system
proteins withoit tryptic proteins within the right mass window missed
80
what are the main steps of sample prep for protein analysis
sample prep: protein extraction, estimation and purification immunprecipatiation, trypsin digest and clean up
81
what is used for data base matching
mascot and GPM
82
what does peakable tables show you
the common proteins detected, I and relative abundance
83
what is a disadvantage of mascot
can't compare 2 different samples
84
why do post translational events happen to proteins and what are they associated with
it modifies the solubility so can move around the cell. Aoosciated with diseases like Parkinson's
85
what is the definition of LC
to separate complex protein mixture into simple ordered system
86
why are sample blanks used in proteomics
sample blanks help you see instrumental noise and background contamination
87
what controls are needed
trypinization and quality
88
what do you use for tyrptinizaiton control
bovine serum albumin
89
why do we freeze thaw
to stop degradation of samples
90
what do you need to be careful about when freeze thawing
keeping cycles the same and as minimum as possible
91
what is the gold standard for discovery work
label free quantification- mass spec based
92
how would you do single protein characterisation
iTRAQ
93
what is silac?
the cells are gown in normal and heavy isotope aa
94
what are the advantages of silac
the isotopically labelled peptides are almost chemically identical
the different samples are mixed at a early step during sample prep
95
what are the disadvantages of silac
- labelled aa might be metabolised to other aa
-expesnove
-not for primary tissue
-increases complexity
-some cells dont grow well
96
what is HELA and what does it give you
shotgun proteomics
total ion chromatogram
base peak chromatogram
ion map- gives a 3d view
97
what is the limit of variation for proteomics
5-10%
98
what is the limit of variation for metabolimics
1-15%
99
what would you use if you want to see the proteins in your sample
global proteome Machine or mascot
100
what is M1 tolerenacing and what is MS2
ms1= 10 ppm
ms2- 25ppm
101
what data file type would you use in mascot
mgf
102
what is a good coverage from mascot
40-100%
