comparison of the quantitative measurement of albumin in

Comparison of the Quantitative Measurement of Albumin in Human Plasma Using ELISA and uHPLC-High Resolution Mass SpectrometryAuthors: Li Liu, Xianglin Yuan, Larry M. Mallis, Charles Saginario, Chu Zhang, and Zhongping (John) Lin

CONCLUSIONS

REFERENCES

Results: We compared the difference between the colorimetric assay of total HSA vis-à-vis the quantitative analysis of HSA using uHPLC coupled to HRMS of the intact proteins.3 Differences between sample analysis using the colorimetric assay versus the uHPLC-HRMS assay were observed and are attributed to the speciation of the different albumins chromatographically. Generally, the native form of HSA attributed to approximately 1/3 of the total albumin in the plasma.

Conclusions: With this method a complete picture of the albumins present in human plasma can be determined, i.e., total albumin as compared to different albumin types (oxidation states, glycation forms, etc.). Differences between the colorimetric assay of total HSA and the quantitative analysis of whole HSA protein types, using uHPLC-HRMS, were observed providing a more complete picture of potential health issues.

HPLC Conditions Description

HPLC Shimadzu LC20AD

Mobile Phase A 0.1% Formic acid in H2O

Mobile Phase B 0.1% Formic acid in AcN

Autosampler Temp Ambient

Separation Type Gradient

Column Waters C4 Column,2.1 x 50 mm, 3.5 µm

Flow Rate 0.50 mL/min

Injection Volume 10 µL

Sciex TT 6600 ESI

Ionization Mode Positive

Assay Range 1 – 200 µg/mL

Sample Treatment PBS Dilution

Anticoagulant Na Heparin

Time (Minutes)

Flow Rate (mL/min)

%B

0 0.5 200.5 0.5 252.0 0.5 802.5 0.5 953.5 0.5 953.6 0.5 205.6 0.5 20

Introduction: Human serum albumin (HSA) is the most abundant protein in human blood plasma. It is comprised of 585 residues with 17 disulfide bonds. Cys34 is its single free thiol group which is thought to act as an oxidant scavenger. Previous chromatographic studies have shown that the HSA-SH fraction of HSA decreases with age; 76% vs. 48%, young vs elderly healthy male subjects, respectively.1,2 The lower percent of SH present is a measure of the frailty of the person. Since ELISA can only measure total albumin, it does not provide a complete assessment of the person’s overall health. Although typical serum albumin levels range from 35-50 mg/mL of serum, lower or higher levels of certain serum albumins indicate disease states (e.g., Cirrhosis of the liver, severe dehydration-kidney disease) which requires treatment. Thus test kits that simply measure the total amount of HSA present in blood plasma may not completely describe the patient condition regarding a measure of frailty of the patient. This poster presents the ability of using LC coupled to high resolution mass spectrometry (HRMS) for the analysis of the different intact albumin proteins.

Method: A sensitive colorimetric assay kit from Sigma-Aldrich, the Bromocresol Green Assay kit (catalog# MAK124), was used to quantitate albumin levels in human sodium heparin plasma samples. The method involves addition of single working reagent to standards, controls and samples in a 96-well polystyrene plate and incubation for 5 minutes at ambient temperature. Following incubation, the plate is read on a plate reader (e.g. SpectraMaxM2e) at 620 nm. The kit utilizes bromocresol green, which forms a colored complex specifically when bound to albumin. The intensity of color (measured at 620 nm) is directly proportional to the albumin concentration in sample. By comparison, a uHPLC-HRMS method which determines the concentration and intact molecular weight of the various whole albumin proteins was used. The advantage of the LC-MS system is the ability to separate different Albumin species present in the plasma.

INTRODUCTION

METHOD

HPLC & HR-MS ConditionsMS Conditions Description

Mass Spectrometer Sciex TT 6600

Ion Source ESI

CUR 50

GS1 50

GS2 50

IS 4500

TEM 500 oC

DP 200

Sample Preparation for BCG Assay Kit

Starting from a 250 mg/mL solution of HAS in DI water, dilute using PBS buffer into a curve range of 150 – 15,000 µg/mL Albumin concentration

Prepare QCs at the LLOQ (150 µg/mL), LQC (450 µg/mL), MQC (7,500 µg/mL), HQC (12,000 µg/mL), and ULOQ (15,000 µg/mL) for run acceptance.

Pre-dilute samples 1:10 by adding PBS for example 20 µL sample + 180 µL of PBS.

Transfer 50 µL of standards, controls, blank, and pre-diluted samples from tubes to 96-well polystyrene plate in duplicate as per plate map.

Add 200 µL of reagent and tap lightly to mix, avoiding bubbles.

Incubate 5 minutes at RT and measure absorbance at 620 nm on plate reader.

SpectraMax M2e ROM V3.022

1 S. Era, T. Hamaguchi, M. Sogami, K. Kuwata, E. Suzuki, K. Miura, K. Kawai, Y. Kitazawa, H. Okabe, A. Noma, et al., Int. J. Pept. Protein Res. 31 (1988) 435-442.2 S. Era, K. Kuwata, H. Imai, K. Nakamura, T. Hayashi, M. Sogami, Biochim. Biophys. Acta 1247 (1995) 12-16.3 L. Turell, H. Botti, L. Bonilla, M. Torres, F. Schopfer, B. Freeman, L. Armas, A. Ricciardi, B. Alvarez, R. Radi. J. Chrom. B. 944 (2014) 144-151.4 M. Domenicali, M. Baldassarre, F. Giannone, M. Naldi, M. Mastroroberto, M. Biselli, M. Laggetta, D. Patrono, C. Bertucci, M. Bernardi, and P. Caraceni, Hepatology. 60:6 (2014) 1851-1860.5 http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/2/mak124bul.pdf

y = 0.0166x - 0.0193R² = 0.9963

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 20 40 60 80 100 120

HSA Native Concentration Curve (Myoglobin IS)

900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

m/z, Da

0.0e0

1.0e3

2.0e3

3.0e3

4.0e3

5.0e3

6.0e3

7.0e3

8.0e3

9.0e3

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

Inte

nsi

ty,

cp

s

*942.6265

*998.0166

*893.0853

*1060.3336

*1130.9716

*1211.6966

*1304.8478

*1413.5248

*1541.9412

*1696.0499

*1884.3948

*2119.9009

+TOF MS from STD3.wiff (sample 1) - STD3, Experiment 1 @ RT: 1.52 from 1.46 to 1.58 min

900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

m/z, Da

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

Inte

nsi

ty,

cp

s

*1280.9530 (1)*1359.3206 (1)*1233.5518 (1)

*1387.6179 (1)

*1447.9036

*1211.1508

*1231.3548*1480.0644

*1189.5341 (1)

*1513.6743*1168.6828

*1548.8619

*1187.4185 (1)

*1585.7166*1148.5483

*1166.6030 (1)

*1624.3667

*1146.5047 (1)*1664.9548

*1129.1030*1060.4274

*998.1154

*1707.6267

*1752.5351

*942.7240

*1799.8780

*1849.8514

*893.1663*1902.6860

1958.6130 (1)

*2017.9562

*2080.9772

*2148.1275

*2144.2886

2219.6676 (1)


900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

m/z, Da

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

Inte

nsi

ty,

cp

s

*998.0735*942.6820

*1060.3923

*893.1324

*1131.0250

*1211.7663 (1) *1280.9566 (1)

*1359.3161 (1)

*1417.1219 (1)

*1447.9044

*1480.0605

*1548.8607

*1585.7244

*1624.3761

*1664.9639

*1707.6264

*1752.5453

*1799.8908

*1849.8634

*1902.7001

*1884.4298 (1)

1958.6466 (1)

2017.9805 (1)

*2119.8808

*2148.1705

*2149.4507


900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

m/z, Da

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

17000

18000

19000

Inte

nsi

ty,

cp

s

*942.6568*998.0474

*893.1131

*1060.3636

*1131.0014

*1211.7335

*1304.8560

*1280.9587 (1)

*1359.3280 (1)

*1387.6245 (1)

*1447.9152 (1)

*1480.0676

*1541.9604

*1548.8815

*1696.0725

*1707.6358

*1884.4286*1752.5511

*1902.7032*1753.9540

*2119.8730*1958.6343


900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

m/z, Da

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

17000

18000

19000

20000

21000

22000

23000

24000

25000

26000

Inte

nsi

ty,

cp

s

*942.6362

*998.0260

*893.0944

*1060.3444

*1130.9811

*1211.7111

*1304.8500

*1413.5290 (1)

*1541.9503

*1696.0586*1306.2820

*1480.0629

*1884.4412*1548.8931

*1624.3878

*2119.85921707.6546 (1)


6.61e4 6.62e4 6.63e4 6.64e4 6.65e4 6.66e4 6.67e4 6.68e4 6.69e4 6.70e4 6.71e4 6.72e4 6.73e4 6.74e4 6.75e4 6.76e4 6.77e4 6.78e4 6.79e4

Mass, Da

0.0e0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

2.6e5

2.7e5

2.8e5

2.9e5

3.0e5

3.1e5

3.2e5

Re

co

nst

ruc

ted

In

ten

sity

67800.70

67899.73

66566.70

Reconstruction from STD4.wiff (sample 1) - STD4, Experiment 1 @ RT: 1.52 from 1.46 to 1.58 min


Mass, Da

0.0e0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

2.6e5

2.7e5

2.8e5

2.9e5

3.0e5

3.1e5

3.2e5

3.3e5

Re

co

nst

ruc

ted

In

ten

sity

67800.26

67899.46



Mass, Da

0.0e0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

Re

co

nst

ruc

ted

In

ten

sity

66559.11

66445.33

66715.55

66725.45

66619.10

67805.60

66879.6966354.09 66792.52

66256.35



Mass, Da

0.0e0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

Re

co

nst

ruc

ted

In

ten

sity

66559.82

66445.60

67804.11

66721.44

66617.80

66794.57 66873.34

66890.2066355.18



Mass, Da

0.0e0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

Re

co

nst

ruc

ted

In

ten

sity

67802.62

66560.80

66446.15

66719.47

66732.02

66616.14

67898.2866793.85 66871.71

66888.97



Mass, Da

0.0e0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

2.6e5

2.7e5

Re

co

nst

ruc

ted

In

ten

sity

67801.21

66562.07

66447.00

67900.9666725.32


900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

m/z, Da

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

17000

18000

19000

20000

21000

22000

23000

24000

25000

26000

27000

28000

29000

Inte

nsi

ty,

cp

s

*942.6299*998.0223

*893.0898

*1060.3391

*1130.9764

*1211.7036

*1304.8523

*1413.5296

*1541.9513

*1696.0569

*1884.3816

*1306.1017*2119.8888


6.45e4 6.50e4 6.55e4 6.60e4 6.65e4 6.70e4 6.75e4 6.80e4 6.85e4 6.90e4 6.95e4

Mass, Da

0.0e0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

Recon

structe

d Inten

sity

66559.6366442.10

66720.06

64848.85

64861.3864913.83 65054.98

Reconstruction from 21Jun16_Human Plasma.wiff (sample 1) - Human Plasma, Experiment 1 @ RT: 1.51 from 1.50 to 1.53 min

A B

C

D

E

Peak ID MW (Da) Isoforms Functional Consequence 4

A 66253 (-187) HSA – DA Loss of chelating function, loss of free radical scavenging activity

B 66343 (-97) HSA + Cysteine - DA Impaired chelating function, loss of free radical scavenging activity

C 66439 Native Form of HSA Native Form of HSA with fully preserved activity

D 66560 (+120) HSA + CYS (+O2) Loss of antioxidant activity

E 66720 (+280) HSA + CYS + GLY Impaired antioxidant and binding activity, conformational alteration

16600 16800 17000 17200 17400

Mass, Da

0.0e0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

Re

co

nst

ruc

ted

In

ten

sity

16951.13

Reconstruction from STD8.wiff (sample 1) - ST...xperiment 1 @ RT: 1.52 from 1.46 to 1.58 min

16600 16800 17000 17200 17400

Mass, Da

0.0e0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

Re

co

nst

ruc

ted

In

ten

sity

16951.13


16600 16800 17000 17200 17400

Mass, Da

0.0e0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

Re

co

nst

ruc

ted

In

ten

sity

16951.13


16600 16800 17000 17200 17400

Mass, Da

0.0e0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

Re

co

nst

ruc

ted

In

ten

sity

16951.13


16600 16800 17000 17200 17400

Mass, Da

0.0e0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

Re

co

nst

ruc

ted

In

ten

sity

16951.13


Mass Spectrum m/z 600-4000Deconvoluted Mass Spectrum

10000 – 68,000 Da200 µg/mL total

100 µg/mL total

50 µg/mL total

20 µg/mL total

10 µg/mL total

5 µg/mL total

HSA Structure

Myoglobin Structure

Typical BCG Albumin Assay Curve

Typical LC-HRMS Albumin Curve

Description of Albumin Peaks Observed by LC-HRMS

16600 16800 17000 17200 17400

Mass, Da

0.0e0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

Re

co

nst

ruc

ted

In

ten

sity

16951.20


http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/2/mak124bul.pdf

comparison of the quantitative measurement of albumin in

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