Protein Estimation Methods - University of Minnesota · Protein Estimation Methods Stephen Harvey August 2016 . Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280

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Protein Estimation Methods

Stephen Harvey

August 2016



Protein in Biological Fluids

Plasma

60 to 80mg/mL

CSF

0.15 to 0.6mg/mL

Urine

0 to 0.8mg/mL healthy

2.O to 20mg/mL mild disease

>100mg/mL chronic kidney disease

BALF

<100µg/mL



Mass Spectrometry

• MALDI and Q-TOF

60,000 Da protein requires 1.0mg/mL

• Triple Quadrople

<10 pg on column (less than ng/mL)

• ITRAQ – Peptides labeled

Requires 40 µg for each aliquot



Dynamic range and Variability of proteins

Plasma

Serum albumin (55% of all)

Interleukin 6, 0 to 5 pg/mL

Urine

Uromodulin (30% n-glycosylated)



Sample preparation

• Concentrate/dilute sample

• Purification • Buffers and high salt

• Detergents for integral protein

• Immunoprecipation (IP) • Detergents throughout procedure

• Gel electrophoresis



IP methods - points of concern

Lysis buffers - Tris, SDS, Triton x-100, EDTA

Dilution buffer - Tris, SDS, Triton x-100, EDTA

Wash buffers - Tris, SDS, Triton x-100, EDTA

Elution buffer - Tris, SDS, DTT



Total protein concentration

• Ultraviolet absorption

• Colorimetric assays

Bradford

Bicinchoninic Acid assay (BCA)



Ultraviolet Absorption

• Proteins Absorbance at 280nm:

Tryptophan, Tyrosine

Absorbance at 205nm:

Peptide bond, sensitive, more interference

• Peptides Absorbance between 200-230nm: Aromatic A.A., histidine, cysteine, methionine



Considerations

Over 10-fold range in UV absorption at 280nm

Extinction coeff: e = ABS/concentration (L)

Varies with pH and ionic strength

• Strongly interfering compounds.

Nucleic acids - measure at 260 and 280nm

If OD260/OD280 < 1.0, ~95% protein

[protein] = 1.55A280 – 0.76A260 (ref. below)

Buffers - especially at 205nm

Detergents - especially at 205nm

Unsaturated compounds - 205nm



Considerations con’t

• Loss to cuvette walls

Dilute solutions measured at 205nm

Stoscheck, C, Methods in Enzymology, Vol 182, pg 54-56



Colorimetric Assays

Bicinchoninic Acid assay (BCA) • 20 to 2000µg/mL

• Cu2+ complexes with C, W, Y and peptide bonds

• Reduced to Cu+

• Cu+ reacts with BCA reagent:

• Absorbance at 562nm

Coomassie Blue Protein Assay (Bradford) • 1.25 to 1400µg/mL

• Basic and aromatic residues stabilizes anionic form of dye.

• Peptides of over 10 residues

• Absorbance at 595nm



Considerations

• Determine linear range.

• Use similar standard to sample

• Determine concentration from standard curve

• Bradford - High protein to protein variability Sensitive to interfering compounds

• BCA is less sensitive to interfering compound.

• Microassays using these reagents are particularly sensitive to interfering agent (up 40-fold more).



Bradford (BioRad) and BCA (Pierce) standard curves 1.0mg/mL bovine serum albumin used as standard

Bradford assay BCA assay

1 to 4µg/mL 5 to 20µg/mL



Assay Conc .(point) Conc. (slope) Bradford(low) not detected ND (high) 0.05mg/mL ND BCA (low) 0.17mg/mL 0.18mg/mL (high) 0.64mg/mL NA UV280nm(high) 0.75mg/mL NA UV280nm(Standard) 1.02mg/mL NA

Uromodulin concentration measure 1.0mg/mL BSA used as standard



Compatible with protein assay

Compound Bradford BCA 280nm/205nm

Triton X-100 0.1% 1.0% 0.02%/<0.01%

Tween 20 0.01% 1.0% 0.3%/0.1%

CHAPS 10% 1.0% 1.0%/1.0%

SDS 0.1% 1.0% 0.1%/0.1%

Octyl-B-glucoside 0.5% 5.0% 10%/ND

Tris 2.0 M 0.1 M 0.5 M/40mM

Phosphate 1.0 M 2 M 1.0 M/50mM

Sodium chloride 5.0 M 1.0 M >1.0 M/0.6 M



Compatible with MS analysis

Compound Bradford BCA LC-MS

Triton X-100 0.1% 1.0% not

Tween 20 0.01% 1.0% not

CHAPS 10% 1.0% SDS-PAGE

SDS 0.1% 1.0% 0.1%SDS-PAGE

Octyl-B-glucoside 0.5% 5.0% yes

Tris 2.0 M 0.1 M not

Phosphate 1.0 M 2 M not

Sodium chloride 5.0 M 1.0 M not



Salts Polymers Detergents

• Complicate interpretation

• Compete with charged ions

• Interfere with MALDI matrix

• Signal suppression

• Contaminate the instrument



McTavish_20150308_sample2_PAX #1 RT: 0.00 AV: 1 NL: 2.97E7T: FTMS + p ESI Full ms [150.00-2000.00]

840 845 850 855 860 865 870 875 880 885 890 895 900

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

854.33990

855.34308

856.34613 892.29700 899.39789876.32074

893.29816877.32672857.34955 894.30225

885.37683

m/z (H+) 854.3382

10mg/mL in methanol

~10,000ppm

Example contaminant affects on mass

spectrometry analysis

+H

+Na +K +2Na

SALTS • Complicate interpretation



McTavish_201503011_MeOH_1_10000B_PAX #1 RT: 0.00 AV: 1 NL: 1.22E8T: FTMS + p ESI Full ms [150.00-2000.00]

840 845 850 855 860 865 870 875 880 885 890 895 900

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

876.32172

877.32501

892.29535

893.29895

878.32825

894.29980

854.33990 879.33240858.71478 895.30017886.74792851.70947841.72809 869.70319 873.71393865.62347

0.001mg/mL

McTavish_20150308_1_100_PAX_150310152526 #1 RT: 0.00 AV: 1 NL: 4.25E8T: FTMS + p ESI Full ms [150.00-2000.00]

845 850 855 860 865 870 875 880 885 890 895 900

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

876.32233

892.29578

877.32526

893.29919

854.34021

855.34375 894.29974878.32837

873.81531856.34650 895.30017879.33136

896.30414857.34912 884.30920871.36804865.82996853.56433846.56982

0.1mg/mL

Diluted in Sigma Optima grade

Methanol >2ppm sodium

>1ppm potassium

~100ppm

+H

+Na

+K

+H

+Na

+K

~1.0ppm



McTavish_20150308_MSMS_1_100_PAX #1 RT: 0.00 AV: 1 NL: 2.01E8T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00]

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

308.09

591.22 876.32

531.20

398.24

892.30

509.22 609.16327.16 559.52281.15

587.55

240.10 409.16 469.18165.07 625.13 832.27387.18 710.23685.44 770.25 909.66

865.63

998.38953.68

• Expected fragment peaks are present complexed with sodium (+23,).

Fragmentation of compound.

(285.1061, +Na)

(853.336, +Na) (568.237, +Na)

(508.216, +Na)



• Salts present in sample alter observed m/z.

• Salt adducts may produce different ion signal response.

• Salt adduct are present with fragmentation. Complicating

identification.



McTavish_20150313_60ng_uLPL_1000pg_uLPAX #1 RT: 0.00 AV: 1 NL: 1.92E8T: FTMS + p ESI Full ms [150.00-2000.00]

500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

714.42

692.41

736.43

747.44681.40

670.39

758.45659.39

769.45

930.22780.46

915.54886.19

871.52648.38

813.48 944.90

974.25

856.84637.77

1003.93827.49

1018.61620.16

1032.95611.35

1047.62

602.75 1077.31

1091.99 1273.77 1339.81585.14 1405.851229.741150.69 1449.88567.33

McTavish_20150313_60ng_uLPL_1000pg_uLPAX #621-841 RT: 1.44-1.95 AV: 221 NL: 8.09E7T: FTMS + p ESI Full ms [150.00-2000.00]

850 855 860 865 870 875 880 885 890 895 900 905

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

125

Re

lative

Ab

un

da

nce

900.87019

886.19502871.51908

856.84363

876.32010

901.87067

857.75600

887.19683

872.52118

868.51132 877.32187

890.52063

906.19097862.16660

858.50942

893.54071

855.80914

864.19145

885.15996

880.26982

899.83536

850.48979 874.50116

866.51451

883.51094

904.18672

895.86616

888.02197

853.75648

Compound w/Na+

+3

+2

+4

+5

Polymers – Polyethylene glycol



Triton X-100 After cleanup

octylglucoside After cleanup

octylglucoside Before cleanup

Yeung,Y, Nieves,E, Angeletti, R, Stanley, E Anal Biochem. 2008 Nov. 15; 382(2): 135-137

Detergents • Signal suppression




Polyethylene Glycol and detergents

• Complicate interpretation

• Compete with charged ions

• Interfere with MALDI matrix

• Signal suppression


~44 m/z Ethylene oxide polymer



Conclusions

Develop purification/enrichment techniques with both protein level assay and mass spectrometry compatibility in mind.

• Ultraviolet spectroscopy

280nm proteins: 200 - 230nm proteins or peptides

Differential absorption at 280nm, determine extinction coefficient

Sensitive measure at 205nm, many interfering compounds



Conclusions con’t

• Colorimetric assays

Dye-based, Bradford

Cu+2 based, BCA Variable protein-to-protein sensitivity

Interfering compounds

• Mass spectrometry

picogram to microgram quantities required

Signal suppression Detergents

Non volatile salts

Etc.



Compound Bradford BCA UV280nm/205nm Acetate 0.6 M 0.2M 0.1/0.01 Acetone 10% 10% Ammonium sulfate 1.0 M 1.5M 50%/9% Ampholytes pH 3–10 0.5% BES 2.5 M Brij 35 5.0% 1%/1% CHAPS 10% 1.0% 1%/1% Citrate, 50 mM <1.0mM 5%/<10mM Deoxycholate 0.1% 5% 0.3%/0.1% DNA/RNA 10.25mg 0.1mg/1.0µg DMSO 5% 5% 20%/<10% DTT 1.0 M <1mM 3mM/0.1mM

Compatible concentrations for protein assays



Compound Bradford BCA UV280nm/205nm EDTA 0.2 M 10mM 30mM/0.2mM EGTA 50 mM Glycerol 99% 10% 40%/5% Glycine 0.1 M 1.0M 1.0M/5.0mM Guanidine-HCl 2.0 M 4.0M HEPES 0.1 M 0.1M ND/<20mM β-Mercaptoethanol, 1.0 M 0.01% 10mM/<10mM MES 0.7 M 0.1 M Methanol 10% 10% MOPS 0.2 M 0.1mM NP-40 0.25% 5.0% Octyl-B-glucoside 0.5% 5.0% 10%/ND OTG 1.0% 5.0%



Compound Bradford BCA UV280nm/205nm Phosphate 1.0 M 2 M 1.0 M/50mM PIPES 0.5 M 0.5 M Potassium chloride 1.0 M <10mM 100mM/50mM SDS 0.1% 1.0% 0.1%/0.1% Sodium chloride 5.0 M 1.0 M >1.0 M/0.6 M Sucrose 1.0 M 40% 2.0 M//0.5 M Tris 2.0 M 0.1 M 0.5 M/40mM Triton X-100 0.1% 1.0% 0.02%/<0.01% Tween 20 0.01% 1.0% 0.3%/0.1% Urea 6.0 M 3.0 M >1.0 M/<0.1 M Stoscheck, C, Methods in Enzymology, Vol 182, pg 52-53 Pierce, ref 1296.2 BioRad Laboratories Inc, technical note 1069



LC-MS compatible detergents

PPS Silent Surfactant http://shop.expedeon.com/products/18-Protein-Solubility/ 129-PPS-Silent-Surfactant/

Big CHAPS deoxy http://www.emdmillipore.com/life-science-research/big-chap-deoxy/ EMD_BIO-256455/p_Ltab.s1L_.8AAAEWhmEfVhTm?PortalCatalogID= merck4biosciences&CountryName=United+States+of+America

ASB series http://www.emdmillipore.com/life-science-research/asb-16 /EMD_BIO-182755/p_hmmb.s1L_.4AAAEWhmEfVhTm

RapiGest SF http://wwwp1.waters.com/webassets/cms/support/docs/715000122.pdf ProteaseMax http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20 Bulletins/101/ProteaseMAX%20Surfactant%20Trypsin%20Enhancer.pdf N-octyl-B-glucopyranoside Sodium Deoxycholate

http://shop.expedeon.com/products/18-Protein-Solubility/





http://www.emdmillipore.com/life-science-research/big-chap-deoxy/









http://www.emdmillipore.com/life-science-research/asb-16







http://wwwp1.waters.com/webassets/cms/support/docs/715000122.pdf

http://wwwp1.waters.com/webassets/cms/support/docs/715000122.pdf

http://www.promega.com/~/media/Files/Resources/Protocols/Technical

http://www.promega.com/~/media/Files/Resources/Protocols/Technical



LC-MS incompatible detergents

Trition X-100

NP-40*

Igepal

Bri-35

Tween-20

Octyl-B-thioglucopyranoside

SDS*

CHAPS*

* May be removed by SDS-PAGE



Reverse-Phase C18 analysis of plasma

WendtPlasmaCOPDHIV_Sample_30b 11/11/14 12:25:05

RT: 0.00 - 19.01

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

10.251.95

1.85 10.209.41

10.37

10.459.5710.47

10.50

11.4811.628.92

0.64

0.518.512.56

0.69 8.091.10

10.57 13.171.42 7.737.192.03

11.276.26 17.0511.832.70 5.88 12.875.50 6.66 17.283.89 13.392.98 14.155.324.24 17.7014.34 18.330.37 16.6816.3714.81

NL:

2.01E10

TIC MS

WendtPlas

maCOPDHI

V_Sample_

30b

WendtPlasmaCOPDHIV_Sample_30b #440 RT: 1.96 AV: 1 NL: 2.82E9

T: FTMS + p ESI Full ms [70.00-1050.00]

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050

m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

188.07034

205.09685

216.13373

146.05986261.14401

169.12506

100.07597 391.23276313.21176

232.15399

420.22290 461.25861 703.57111639.79681514.00696 556.42462

Analytes present at 1.96 minutes

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Protein Estimation Methods - University of Minnesota · Protein Estimation Methods Stephen Harvey August 2016 . Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280