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analytical exam 2 > proteomics > Flashcards

proteomics Flashcards

1
Q

how many aa are there?

A

20

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2
Q

which aa is named after silk

A

serine

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3
Q

which aa is pro

A

proline- contraction of pyrrolidine

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4
Q

what amino acid is names after roots of plants?

A

valine

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5
Q

what aa is named after the colour white

A

leucine

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6
Q

which aa is gln

A

glutamine

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7
Q

which aa is named after a structurally similar aa

A

isoleucine

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8
Q

which aa was first found in old cheese

A

tyrosine

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9
Q

which aa is named after bladder?

A

tyrosine

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10
Q

which aa is met

A

methionine

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11
Q

what are the two aa ending in acid?

A

aspartic acid and glutamic acid

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12
Q

which aa is named after juice?

A

asparagine

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13
Q

which aa was hypothesised to be similar to another?

A

glutamine

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14
Q

what are peptides held together by

A

peptide bonds

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15
Q

how many aa are a helix

A

10

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16
Q

how many aa in beta pleated sheet

A

6

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17
Q

what is the size of phenylalanine hydroxylase

A

108kDA

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18
Q

in 2 ul of plasma how much protein is there

A

1060 unique proteins

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19
Q

how big is a growth hormone

A

22 kDa

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20
Q

How big is ferritin

A

42 kDa

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21
Q

how big is a single ferritin unit

A

480 kDa

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22
Q

which direction does the b ions go from

A

left to right

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23
Q

what direction does the y ion go from

A

right to left

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24
Q

how are b and y ions made

A

electrospray ionisation

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25
Q

why do we need high reolsultion for mass spec

A

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

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26
Q

what is mass resolution

A

ability to resolve closely adjacent peaks

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27
Q

what is the advantage of madli matrix

A

can analyse high masses so big proteins can lookout changes in proteins in transit modifications as can look at the whole protein

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28
Q

what are the disadvantages of maldi

A

can only look at one protein at a time and so has limited throughput doesn’t use MS2 so have to caluclate mass

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29
Q

how many common proteins are found in the blood?

A

289

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30
Q

how would you analyse complex mixtures

A

would use electrophoresis to break it down into fragments so easier to analyse.

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31
Q

how many proteins detected in the human proteome atlas

A

30k

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32
Q

how many peptides are detected in the human proteome atlas

A

290k

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33
Q

what are the methods for protein estimation

A

-bradord assay
-lowry method
-bicinchronic acid assay
-flourscent based assay
all rely on liaise

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34
Q

what is the sample prep for proteomic analysis

A

protein extraction, estimation and purification immunoprecipiatation, trypsin digestion and clean up

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35
Q

which mass anaylzer has the highest relation

A

orbi trap

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36
Q

what is mass resolution

A

the ability to resolved closely related adjunct mass peak. The bigger the number the higher the mass resolution

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37
Q

what is scan speed

A

how fast the mass analyser scan the entire range of mass spec

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38
Q

which modes would you use on mass spec if you were looking for specific peptides

A

MRN and PRM

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39
Q

what mass spec mode would you use for discovery work (identifying as many polypeptides as possible)

A

DDA and DIA

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40
Q

How does DDA work?

A

relys on chromatography to separate complex mixtures and elutes the most abundant- easier to use for discovery work

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41
Q

how does DIA work

A

splits actual space into blocks and fragments everything has higher fidelity

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42
Q

what do we need to do for mass spec

A

they look at protein mixtures so need to unfold protein and typsinise them

43
Q

what are the advantages of madli

A

looks at intact proteins. Can look at big proteins as has a high mass range

44
Q

what is the disadvtange of madli

A

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
Q

how does MALDI work

A

laser burns through the matrix and into sample but make sure doesn’t degrade samples. It ionises and the ions travel down TOF.

46
Q

how many proteins have been identified in the human body

A

30k

47
Q

what can you do to complex mixtures to make it more basic

A

can fractionate mixtures into crude molecular sizes by gel electrophoresis

48
Q

what is the limitation of proteomics

A

can only identify 60-80% of proteins as they will fall below detection

49
Q

how many peptide sequences are in the body?

A

290k

50
Q

what lipase techniques are used for protein estimation

A

-lowry method
-bradford assay
-bicinchronic acid assay
-fluoroscent based assay

51
Q

what do you want to know before testing a sample

A

a rough estimate of protein in the sample so taht you know how much topspin to include

52
Q

how do you do a rough estimate of protein in a sample

A

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
Q

what is the ratio of tryspsin you want to use

A

20-50:1

54
Q

what is the gel base fractoination method

A

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
Q

what are steps of shot gyn proteomic analysis

A

-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
Q

what does shot gun proteomics tell you

A

won’t know which fragment belongs to which protein. instead can see peptide structures being eluted so know method is working

57
Q

what is the optimum temp for trypsin digestion

A

37 degrees

58
Q

how does trypsiin work

A

cleaves the C term of the K R unless next to proline

59
Q

what do you have to do to sample before trypsin

A

denture the protein in 6-8 M of urea

60
Q

what do you do with trypsin digestion

A

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
Q

what is reposnivle for seperation in a highly reproducible manner

A

LC front end

62
Q

what is included to allow mass measurement and determination

A

electric/magnetic field, vacuum, super shaped electrodes and high speed switching micro electronics

63
Q

how is LC chromotographic done

A

analytical LC proteomic column and solvent gradient profile

64
Q

what are the results from a mass analyser

A

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
Q

explain the electro spray ionisation of the analyte

A

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
Q

what is in silco

A

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
Q

what are the advantages of top down proteomics

A
  • 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
Q

what are the disadvantages of top down proteomics

A

-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
Q

what is the advantages of bottom up proteomics

A

-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
Q

what is are examples of bottom up proteomics

A

shot gun

71
Q

what is top down proteomics

A

high resolution mass spec - intact protein analysis

72
Q

what are the disadvantages of bottom up

A

-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
Q

What is the definition of proteome

A

Profiling an entire set of proteins within a system/cell at a given time and defined conditions

74
Q

What is a limitation of proteomics

A

60-80% detection

75
Q

How do you increase protein coverage after gel electrophoresis

A

By using LC/MS methodology

76
Q

what do the mass analyses do

A

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
Q

what is the disadvantages of top down/maldi

A

low sensistivity
high sample pruirty needed
operator skill
expensive instruments.

78
Q

what are the advantages of bottom up proteomics

A
  • can analysis more complex mixtures
    higher throughput
    greater level of chemical and bioodiucal function can be obtained
    MS2 information
    greater anyalytical flexibity
79
Q

what are the disadvantages of bottom up

A

high rate of false positive
sample prep
complex system
proteins withoit tryptic proteins within the right mass window missed

79
Q

what are the disadvantages of bottom up

A

high rate of false positive
sample prep
complex system
proteins withoit tryptic proteins within the right mass window missed

80
Q

what are the main steps of sample prep for protein analysis

A

sample prep: protein extraction, estimation and purification immunprecipatiation, trypsin digest and clean up

81
Q

what is used for data base matching

A

mascot and GPM

82
Q

what does peakable tables show you

A

the common proteins detected, I and relative abundance

83
Q

what is a disadvantage of mascot

A

can’t compare 2 different samples

84
Q

why do post translational events happen to proteins and what are they associated with

A

it modifies the solubility so can move around the cell. Aoosciated with diseases like Parkinson’s

85
Q

what is the definition of LC

A

to separate complex protein mixture into simple ordered system

86
Q

why are sample blanks used in proteomics

A

sample blanks help you see instrumental noise and background contamination

87
Q

what controls are needed

A

trypinization and quality

88
Q

what do you use for tyrptinizaiton control

A

bovine serum albumin

89
Q

why do we freeze thaw

A

to stop degradation of samples

90
Q

what do you need to be careful about when freeze thawing

A

keeping cycles the same and as minimum as possible

91
Q

what is the gold standard for discovery work

A

label free quantification- mass spec based

92
Q

how would you do single protein characterisation

A

iTRAQ

93
Q

what is silac?

A

the cells are gown in normal and heavy isotope aa

94
Q

what are the advantages of silac

A

the isotopically labelled peptides are almost chemically identical
the different samples are mixed at a early step during sample prep

95
Q

what are the disadvantages of silac

A
  • labelled aa might be metabolised to other aa
    -expesnove
    -not for primary tissue
    -increases complexity
    -some cells dont grow well
96
Q

what is HELA and what does it give you

A

shotgun proteomics
total ion chromatogram
base peak chromatogram
ion map- gives a 3d view

97
Q

what is the limit of variation for proteomics

A

5-10%

98
Q

what is the limit of variation for metabolimics

A

1-15%

99
Q

what would you use if you want to see the proteins in your sample

A

global proteome Machine or mascot

100
Q

what is M1 tolerenacing and what is MS2

A

ms1= 10 ppm
ms2- 25ppm

101
Q

what data file type would you use in mascot

A

mgf

102
Q

what is a good coverage from mascot

A

40-100%

analytical exam 2 (6 decks)

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