Peptide Concentration Assays
2
BSA Thermo y =783.62x- 26.739 R² =0.9963 0 300 600 0 0.4 0.8 u g / m l A595 BSA Thermo y = 289.93x2 + 548.65x + 4.0429 R² = 1 0 250 500 0 0.4 0.8 u g / m l A562 Coomassie-stained SDS-PAGE gel to verify protein extraction and digestion Determining the Most Accurate Assay for Measuring Peptide Concentra tion Jeneal Carter, Janice L. Hallows, Robert L. Moritz Individual Assays and Results Conclusions Abstract NanoDrop (A 280 ) dotMETRIC BCA (A 562 ) OPA (360/460) Bradford (A 595 ) Pipette 1ul of sample onto reading plate, close arm, and take measurements. Make sample dilutions in HEPES to reach 0.5 mg/ml, then mix with dotMETRIC buffer . Apply 1ul spots to strips in triplicate, using capillaries. Fix and develop, then measure. Add 20ul sample + 200ul OPA reagent in an opaque 96-well plate. Measure fluorescence at excitation 360nm and emission 460nm within 1-5 minutes of mixing. Mix 5ml dye + 25ml MQ H 2 O and filter to make reagent. Add 160ul sample + 40ul reagent in a 96-well plate. Incubate at RT x 5min. Measure absorbance at 595nm. Mix 9000ul A + 180ul B to make working reagent. Add 25ul sample + 200ul WR in a 96-well plate. Mix x 30sec. Cover, incubate at 37 o C x 30min. Cool to RT, measure absorbance at 562nm. A variety of assays are commonly used to determine protein concentration, however, an effective assay to determine peptide concentration has not yet been established. Peptide samples prese nt challenges that protein samples do not: •Peptides typically do not contain the specific amino acid residues (e.g. tyrosine and tryptophan) that are recognized by the assay. - •The standards used for determining protein concentration (e.g. a protein solution of known concentration ) do not perform well with peptides. - In order to determine which assay most accurately measures peptide concentration, we prepared a series of standards (25-500 ug/ml) using 5 different types of starting material: 1. Commercially available BSA solution (Thermo Scientific) 2. BSA solution prepared in the lab (concentra tion determined by A280/extinction coefficient (e)) 3. Whole cell ly sate of th e HCT-15 colo n cancer ce ll line (concentration determined by BCA assay) 4. BSA peptides produced b y trypsin dig estion of the BSA solution (#2 above) 5. HCT-15 peptide s produced by trypsin digestion of the HCT-15 lysate (#3 above) The five protein assays listed in Table 1 were performed using all 5 sets of standards, and the 500 ug/ml standard from each set was arbitrarily chosen to serve as the “unknown” for the assays. lyse cells extract protein reduce alkylate digest peptide standards Assay Method InterferingAgents Calculations Misc. Dynamic Range BCA (A562) Thermo Scientific Colorimetric: Bicinchoni nicacid(BCA)detectsfor Cu+1 (formedwhenCu2+ isreducedby proteinin analkalineenvironment )yieldinga purple- coloredreactionproduct. Asco rbicAc id Catecholamines Creatinine Cysteine EGTA Impure Glycerol Hydrogen Peroxide Hydrazides Iron Lipids Melibiose Phenol Red Impure Sucrose Tryptophan Tyrosine Uric Acids standardcurve detergent compatible fairly stable under alkaline conditions 20-2000ug/ml Bradford (A595) BioRad Colorimetric: Dyeturnsfromreddish/browntoblueas proteinbindstothe coomassie dye inthe acidicenvironmen t of the reagent. Some Detergents Flavonoids SodiumHydroxid e Basic ProteinBuffers standardcurve 2xasmuch protein-to- proteinvariation thanBCA 50-500ug/ml NanoDrop (A280) Thermo Scientific ProteininsolutionabsorbsUVlight at a wavelengt hof280nm, duetothe presence of aromaticaminoacids, mainly tyrosine andtryptophan . Any non-proteincompone nt that absorbs ultraviolet light e.g.: Beer’sLaw (multiply by extinction coefficient) considerable error for protein mixtures 100-100,000 ug/ml Nucleotides NucleicAcids dotMETRIC GBiosciences Diameter of proteinspotsisproportion al totheir proteinconcent ration. Resistant t omost commonlaborato ry agents. measure dots using exponential scale noprotein-to- proteinvariation 0.031-2.0 ug/ml OPA (360/460) Thermo Scientific o-phthala ldehyde(OPA)reactswiththe primary aminesof the proteininthe presence of mercaptoe thanol, yieldinga blue-color edfluorescent product which hasamaximumwavelengthof excitationof 340nmandemissionat 455nm. Amine-cont ainingbuffers linear trend line compatible with detergentsand reducingagents .050 ug/ml- 25ug/ml Figure 2: developed spots A1 0.051 A1 0.039 A1 0.100 A2 0.054 A2 0.039 A2 0.063 A3 0.047 A3 n/a A3 0.044 avg 0.051 avg 0.039 avg 0.069 conc (*6 dil) 0.304 conc (*6 dil) 0.234 conc (*6 dil) 0.414 A1 0.051 A1 A2 0.047 A2 A3 0.054 A3 av g 0. 051 conc (*6 dil) 0.304 1 .1 1 . . . . n o t v i s i b l e i i l JC BS A ( 9.741 ) BSA pe pt ides ( 2) BS A Pie rc e(2) HCT-1 5 ( 10. 11) HCT-1 5 p ept ide s ( 2) . .1 A1 5.913 A1 1.187 A1 1.390 A2 5.960 A2 1.135 A2 1.380 A3 5.936 A3 1.149 A3 1.399 avg 5.936 avg 1.157 avg 1.390 ε 0.667 ε 0.667 ε 0.667 conc 8.900 conc 1.735 conc 2.083 A1 29.617 A1 5.752 A2 29.748 A2 5.253 A3 29.402 A3 5.133 avg 29.589 avg 5.379 . . . . . . JC BS A(9.7 41 ) BSApe pt id es (2 ) BS APie rc e(2) HCT-15 (10 .11 ) HCT-15 pep tides (2) Methods "Unknowns" BSA Thermo JC BSA BSA peptides HCT-15 lysate HCT-15 peptides BSA Thermo (0.5mg/ml) n/a 0.471 0.518 0.444 0.423 JCBSA( 0.5mg/ml) 0.535 n/a 0.549 0.470 0.449 BSA peptides(0.5mg/ml) 0.483 0.450 n/a 0.425 0.404 HC T- 15 ly sat e (0 . 5mg/ ml) 0. 567 0.5 29 0.581 n/ a 0.477 HCT -1 5 pe pti des(0 .5mg/ml) 0.589 0.5 50 0.6 04 0. 517 n/a Standards "Unknowns" BSA T her mo JC B SA BSA p eptides HCT-15 l ysate HCT-15 p eptides BSA Thermo (0. 5mg/ml) n/a 0.501 2.635 0.681 3.263 JC BSA(0.5mg/ml) 0.474 n/a 2.547 0.658 3.152 BSA peptides(0.5mg/ml) 0.067 0.0 42 n/a 0.114 0.5 87 HCT-15 lysate (0.5mg/ml) 0.118 0.098 0.745 n/a 0.913 HCT-15 pept ide s(0 .5mg/ ml) 0. 35 4 0.352 1.937 0.4 97 n/ a Standards BSA Thermo y = 0.0241x + 4.1095 R² = 0.9993 0 250 500 0 10000 20000 u g / m l 360/460 Table 1: Individual protein assays Figure 1: Preparation of peptide standards BSA Th ermo J CB S A BS A pept ides HC T- 15 l ys ate HC T- 15 pepti des BSATher mo (0 .5mg/ml) n/a 0.430 0.598 0.497 0.7 01 JCBSA(0.5mg/ml) 0.577 n/a 0.696 0.571 0.843 BSApeptides(0.5mg/ml) 0.425 0.361 n/a 0.422 0. 5 66 HCT-1 5 lysate (0 .5mg/ml) 0.503 0.433 0.602 n/a 0.707 HCT -1 5 pe pti de s(0 .5 mg/ ml ) 0.3 86 0.3 25 0. 452 0. 383 n/ a Standards "Unknowns" Assay Advantages Disadvantages Works with Peptides? BCA (A562) Easy Good sensitivity for proteins Dependent on type of standard No Bradford (A595) Very quick Not as sensitive No NanoDrop (A280) Works well for individual protein but not complex mixtures Large variability even for singleproteinsolutions No dotMETRIC No interfering agents Low reproducibili ty Tech nically difficult Measuring spots is subjective No OPA Quick Reproducible Not dependent on type of standard used Very sensitive to air bubbles Yes Table 2: Results Funding
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8/12/2019 Peptide Concentration Assays
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BSA Thermo
y =783.62x- 26.739R² =0.9963
0
300
600
0 0.4 0.8
u g / m l
A595
BSA Thermo
y = 289.93x 2 + 548.65x + 4.0429R² = 1
0
250
500
0 0.4 0.8
u g / m l
A562
Coomassie-stained SDS-PAGE gelto verify protein extraction anddigestion
Determining the Most Accurate Assayfor Measuring Peptide Concentration
Jeneal Carter, Janice L. Hallows, Robert L. Moritz
Individual Assays
and Results
Conclusion
Abstract
NanoDrop (A 280 )
dotMETRICBCA (A562 )
OPA (360/460Bradford (A 595 )
Pipette 1ul of sample onto reading plate, close arm, and takemeasurements.
Make sample dilutions in HEPES to reach 0.5 mg/ml, then mix withdotMETRIC buffer. Apply 1ul spots to strips in triplicate, using
capillaries. Fix and develop, then measure.
Add 20ul sample + 200ul OPA reagent in an opaqMeasure fluorescence at excitation 360nm and emwithin 1-5 minutes of mixing.
Mix 5ml dye + 25ml MQ H 2O and filter to make reagent. Add 160ulsample + 40ul reagent in a 96-well plate. Incubate at RT x 5min.Measure absorbance at 595nm.
Mix 9000ul A + 180ul B to make working reagent. Add 25ul sample+ 200ul WR in a 96-well plate. Mix x 30sec. Cover, incubate at 37 oC
x 30min. Cool to RT, measure absorbance at 562nm.
A variety of assays are commonly used to determineprotein concentration, however, an effective assay todetermine peptide concentration has not yet beenestablished. Peptide samples present challenges thatprotein samples do not:
• Peptides typically do not contain the specificamino acid residues (e.g. tyrosine and tryptophan)that are recognized by the assay.-
• The standards used for determining proteinconcentration (e.g. a protein solution of knownconcentration) do not perform well with peptides.-
In order to determine which assay most accuratelymeasures peptide concentration, we prepared a seriesof standards (25-500 ug/ml) using 5 different types ofstarting material:1. Commercially available BSA solution (Thermo
Scientific)2. BSA solution prepared in the lab (concentration
determined by A280/extinction coefficient ( e ))3. Whole cell lysate of the HCT-15 colon cancer cell
line (concentration determined by BCA assay)4. BSA peptides produced by trypsin digestion of the
BSA solution (#2 above)5. HCT-15 peptides produced by trypsin digestion of
the HCT-15 lysate (#3 above)The five protein assays listed in Table 1 wereperformed using all 5 sets of standards, and the 500ug/ml standard from each set was arbitrarily chosen toserve as the “unknown” for the assays.
lyse cells
extract proteinreducealkylatedigest
peptide standards
Assay Method InterferingAgents Calculations Misc. Dynamic RangeBCA (A562)ThermoScientific
Colorimetric:Bicinchoninicacid(BCA)detectsfor Cu +1 (formedwhenCu 2+ isreducedby proteininanalkalineenvironment)yieldinga purple-coloredreactionproduct.
AscorbicAcidCatecholaminesCreatinineCysteineEGTAImpure Glycerol
HydrogenPeroxideHydrazidesIronLipidsMelibiose
Phenol RedImpure SucroseTryptophanTyrosineUric Acids
standardcurve detergentcompatible fairly stableunder alkalineconditions
20-2000ug/ml
Bradford(A595)BioRad
Colorimetric:Dye turnsfrom reddish/browntoblueasproteinbindstothe coomassie dye intheacidicenvironment of the reagent.
Some DetergentsFlavonoidsSodiumHydroxideBasic ProteinBuffers
standardcurve 2xasmuchprotein-to-proteinvariationthanBCA
50-500ug/ml
NanoDrop(A280)ThermoScientific
ProteininsolutionabsorbsUVlight at awavelengthof 280nm, due tothe presenceof aromaticaminoacids, mainly tyrosineandtryptophan.
Any non-proteincompone nt that absorbsultraviolet light e.g.:
Beer’sLaw (multiply byextinctioncoefficient)
considerableerror for proteinmixtures
100-100,000ug/ml
NucleotidesNucleicAcids
dotMETRICGBiosciences
Diameter of proteinspotsisproportionaltotheir proteinconcentration.
Resistant tomost commonlaboratory agents. measure dotsusingexponentialscale
noprotein-to-proteinvariation
0.031-2.0 ug/ml
OPA(360/460)ThermoScientific
o-phthalaldehyde(OPA)reactswiththeprimary aminesof the proteininthepresence of mercaptoethanol, yieldingablue-coloredfluorescent product whichhasamaximumwavelengthof excitationof340nmandemissionat 455nm.
Amine-containingbuffers linear trendline
compatible withdetergentsandreducingagents
.050 ug/ml-25ug/ml
Figure 2: developed spots
A1 0.051 A1 0.039 A1 0.100A2 0.054 A2 0.039 A2 0.063A3 0.047 A3 n/a A3 0.044avg 0.051 avg 0.039 avg 0.069
conc (*6 dil) 0.304 conc (*6 dil) 0.234 conc (*6 dil) 0.414
A1 0.051 A1A2 0.047 A2A3 0.054 A3a vg 0 .0 51
conc (*6 dil) 0.304
1 .1 1...
.
n o t v
i s i b l
e
i i
l
J C B SA (9. 74 1) BS A p ep ti des (2 ) B SA Pi er ce (2 )
H CT-15 (10 .11 ) H CT-15 pep tides (2 )
. .1
A1 5.913 A1 1.187 A1 1.390A2 5.960 A2 1.135 A2 1.380A3 5.936 A3 1.149 A3 1.399avg 5.936 avg 1.157 avg 1.390
ε 0.667 ε 0.667 ε 0.667conc 8.900 conc 1.735 conc 2.083
A1 29.617 A1 5.752A2 29.748 A2 5.253A3 29.402 A3 5.133avg 29.589 avg 5.379
. .
. .
. .
J C B SA (9. 74 1) BS Ap ep ti de s ( 2) B SA Pi er ce (2)
HCT-1 5 (1 0.11) HCT-1 5 p eptides (2 )
Methods
"Unknowns" BSA Thermo JC BSA BSA peptidBSA Thermo (0.5mg/ml) n/a 0.471 0.518
JC BSA (0.5mg/ml) 0.535 n/a 0.549 BSA peptides (0.5mg/ml) 0.483 0.450 n/a
H CT -15 l ysa te ( 0. 5mg/ ml) 0 .567 0. 529 0. 58 1 HC T- 15 p ept ide s( 0. 5mg/ ml ) 0 .589 0. 550 0. 60 4
Standard"Unknowns" BSA Ther mo JC BSA BSA peptides HCT-15 lysate HCT-15 peptides
BSA Thermo (0. 5mg/ml) n/a 0. 501 2.635 0.681 3. 263JC BSA (0.5mg/ml) 0.474 n/a 2.547 0.658 3.152
BSA peptides (0.5mg/ml) 0.067 0. 042 n/a 0.114 0. 587HCT- 15 lysate (0. 5mg/ml) 0.118 0. 098 0.745 n/a 0. 913
HC T- 15 p ep tid es ( 0. 5mg /ml ) 0 .3 54 0. 352 1 .93 7 0. 497 n /a
Standards
BSA Thermo
y = 0.0241x + 4.1095R² = 0.9993
0
250
500
0 10000
u g / m l
360/460
Table 1: Individual protein assays
Figure 1: Preparation of peptide standards
BS A T her mo J CB SA B SA pep ti des H CT -15 ly sa te H CT -15 pe pt ide sBSA The rmo ( 0.5mg/ml) n/a 0.430 0.598 0.497 0. 701
JC BSA (0.5mg/ml) 0.577 n/a 0.696 0.571 0.843BSA peptides (0.5mg/ml) 0.425 0.361 n/a 0.422 0. 566
HCT- 15 lysate ( 0.5mg/ml) 0.503 0.433 0.602 n/a 0. 707HC T- 15 p ept id es ( 0. 5mg /m l) 0. 386 0. 325 0 .45 2 0 .38 3 n /a
Standards"Unknowns"
Assay Advantages Disad
BCA (A562 ) EasyGood sensitivity for proteins
Dependent onstandard
Bradford (A 595 ) Very quick Not as sensitivNanoDrop (A 280 ) Works well for individual
protein but not complexmixtures
Large variabilsingle protein
dotMETRIC No interfering agents Low reproducibiTechnically dMeasuring sp
OPA QuickReproducibleNot dependent on type ofstandard used
Very sensitive
Table 2: Results
Funding
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