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