Quantitative Analysis of Proteins in Human Serum by Species-unspecific Isotope Dilution-gel Electrophoresis-laser Ablation-inductively Coupled Plasma Mass Spectrometry†

doi:10.7503/cjcu20140233

Abstract

Abstract:

A methodology based on species-unspecific isotope dilution-gel electrophoresis-laser ablation coupled with inductively coupled plasma mass spectrometry(GE-LA-ID-ICP-MS) was established for the absolute quantification of proteins in human serum by measuring sulfur. The electrophoresis separation condition of human serum were 7.5% separating gel, 4% stacking gel, 100 V until bromophenol blue dye had migrated to the bottom of gel. The average signal intensity of ³²S at different concentrations of sulfur enriched gels(1—400 μg/g) showed a good linear relationship. The relative quantitative analysis of transferrin and albumin in human serum was investigated using external standard method. In addition, the created species-unspecific isotope dilution in GE-LA-ICP-MS had added ³⁴S spike signals into gels successfully by mixing sulfur spike with protein strips. According to isotope dilution equation, the method was applied to a fast, accurate absolute quantification of transferrin and albumin in human serum. The methodology was validated by comparing with the quantitative analysis of a serum protein certified reference material(ERM-DA470/IFCC). The results confirmed that determined concentration of transferrin and albumin fitted well with the certified value and the methodology had good accuracy and better measurement precision than external standard method.

Key words: Species-unspecific isotope dilution, Laser ablation, Inductively coupled plasma mass spectrometry, Transferrin, Albumin

CLC Number:

O657.63

TrendMD:

ZHANG Dan, FENG Liuxing, WANG Jun, SHEN Dairui, XIONG Jinping. Quantitative Analysis of Proteins in Human Serum by Species-unspecific Isotope Dilution-gel Electrophoresis-laser Ablation-inductively Coupled Plasma Mass Spectrometry^†[J]. Chem. J. Chinese Universities, 2014, 35(9): 1889.

Figures/Tables 8

Fig.1 Calibration curve for sulfur in enriched gels

Fig.2 Calibration curves for transferrin(A) and albumin(B) in serum by GE-LA-ICP-MS

Table 1 Quantitative results of transferrin and albumin in ERM-DA470/IFCC by external calibration GE-LA-ICP-MS

Protein	Standard curve equation	Protein concentration/(mg·mL^-1, n=3)	Certified value/(mg·mL^-1)
Transferrin	y=6791.06x-3109.96	2.63±0.15	2.32±0.08
Albumin	y=6781.35x+12708.99	33.49±3.13	36.5±1.2

Fig.3 LA-ICP-MS profiles obtained for 34S(a), 32S(b) and isotope ratio 32S/34S(c) in transferrin(Tf) through species-unspecific isotope dilution treatment after electrophoresis

Fig.4 LA-ICP-MS profiles obtained for transferring (Tf) in ERM-DA470/IFCC a. 34S; b. 32S; c. isotope ratio of 32S/34S.

Table 2 Quantitative results of transferrin by species-unspecific GE-LA-ID-ICP-MS

Transferrin	Isotope ratio, R_b(³²S/³⁴S, n=3)	m_z/m_y	Protein concentration/(mg·mL^-1)
Standard protein	3.37±0.05	2.04	2.96^*
ERM-DA470/IFCC	2.70±0.06	2.04	2.29±0.05
Human serum sample	2.87±0.07	2.04	2.45±0.06

Fig.5 LA-ICP-MS profiles obtained for albumin(Alb) in ERM-DA470/IFCC a. 34S; b. isotope ratio of 32S/34S; c. 32S.

Table 3 Quantitative results of albumin by species-unspecific GE-LA-ID-ICP-MS

Albumin	Isotope ratio, R_b(³²S/³⁴S, n=3)	m_z/m_y	Protein concentration/(mg·mL^-1)
Standard protein	6.72±0.18	2.17	28.12^*
ERM-DA470/IFCC	7.75±0.15	2.17	34.75±1.02
Human serum sample	8.06±0.22	2.17	36.97±1.55

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