Automated quantitation of total protein in cultured skin fibroblasts

ELSEVIER

Clinica Chimica Acta 259 (1997) 129 136

Automated quantitation of total protein in cultured skin fibroblasts A n d r e w J. Williams a,b, J o h n C. Coakley b, J o h n Christodoulou a'* aDepartmeiTt of Paediatrics and Child Health, University of Sydney, Sydney, N.S.W. 2050, Australia bDepartment of Clinical Biochemistry, Royal Alexandra Hospital for Children, Westmead, N.S.W. 2145, Australia Received 21 May 1996; revised 12 November 1996; accepted 14 November 1996

Abstract

An automated Coomassie Blue method of measuring total protein in cultured skin fibroblasts using a random access analyzer, the Roche MIRA, was compared to a manual dye binding method using bicinchoninic acid. The automated Coomassie Blue method is in common use in many routine laboratories for measurement of total protein in urine and CSF, and was found by us to be significantly faster than a manual protein method, with an average assay time of 2 min per sample. In addition, intra- and inter-run precision were comparable for the automated and manual methods, and compared favourably with other automated methods. We recommend that where the MIRA apparatus is available, consideration be given to its use for protein quantitation of cell or tissue extracts. © 1997 Elsevier Science B.V.

Keywords: Protein quantitation; Automation; Coomassie Blue; Bicinchoninic acid; Fibroblast

1. Introduction

Enzyme measurements in tissues are usually expressed as a ratio of the amount of protein in the sample and traditionally protein has been mea* Corresponding author, University Dept. of Paediatrics and Child Health, Royal Alexandra Hospital for Children, P.O. Box 3515, Parramatta N.S.W. 2124, Australia. Tel.: 612 8453452; fax: 612 8453389; e-mail: [email protected] 0009-8981/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PH S0009-898 1(96)06479-0

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sured by manual methods which depend on the reduction of copper in the presence of a chromogenic chemical, such as the Lowry [1,2] and bicinchoninic acid (BCA) [3] methods. Although reliable, these methods are time consuming and are subject to error in the presence of certain buffer reagents and detergents [1,3]. Since enzyme assays are often expressed as a ratio of the protein concentration, imprecision in this method would be magnifed by any imprecision of the protein assay. Autoanalyzers are capable of fast and precise measurements of many analytes. We have adapted a micro-protein method on the Roche MIRA for measuring total protein in cultured skin fibroblasts, the aim being to develop a rapid and reliable way to quantitate protein levels. We compared the automated method to a manual method of measuring total protein using BCA and present data on precision and effect of possible interfering agents on the new method.

2. Materials and methods

2.1. Fibroblast preparation Skin fibroblasts were grown in 75-cm 2 culture flasks under standard conditions [4]. When confluent, cells were removed from the flask with trypsin-EDTA and washed 3 times with phosphate buffered saline to remove all trace of media. The skin fibroblasts were then disrupted in three sequential steps: (1) Digestion - - Digitonin (1 mg/ml) (Sigma) was added to the cell extract for 30 s [5] which was then centrifuged at 10000 x g for 1 min, the supernatant removed and infranatant washed in 1 ml of MOPS buffer (composed of 20 mmol/1 MOPS [2-N-morpholino-propanesulphonic acid], 3 mmol/l sodium EDTA, 0.25 mol/1 sucrose, pH 7.2), and then resuspended in 400 pl of MOPS buffer. (2) Freeze/thaw - - The washed fibroblasts from the first step were placed in a mix of dry ice and methanol until frozen then in warm water until thawed. This procedure was repeated twice. (3) Sonication - - Six pulses at 30% of duty cycle on a Branson II sonifier (model number B250, Branson Instruments, Danbury, CT, USA). The cell extracts were then frozen for up to 1 month at - 8 0 ° C . The fibroblast extract was thawed and allowed to reach room temperature prior to measurement of total protein.

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2.2. Protein assays 2.2.1. Automated method

Total protein was measured by the Coomassie Blue (CB) method [6] and performed by a random access autoanalyzer, the Cobas MIRA at 37°C. Sample (10 ~1) was added to Coomassie Blue reagent (500 /~1) (Quantimetrix, Hawthorne, CA), mixed and the absorbance at 600 nm was measured after 75 s. The Coomassie Blue dye interacts with amino groups of proteins to form a blue colour which absorbs maximally at 595 nm. Four aqueous protein standards (Quantimetrix) (0.25, 0.5, 0.75 and 1.00 g/l) and a zero standard were used to construct a standard curve and the protein concentration was determined by the autoanalyzer's microprocessor. Specimens with a protein value above 1.0 were automatically diluted by the MIRA to increase the range to 5 g/1. 2.2.2. Manual method

We measured total protein using bicinchoninic acid reagent (Pierce, Sydney, Australia). Cupric ions are reduced by protein to cuprous ions which then bind specifically to BCA molecules [3]. Individual samples, standards and controls (0.5 ml) were added to BCA reagent (2 ml) and incubated at 37°C for 30 min. The mixture was cooled and the absorbance measured at 562 nm in an Ultrospec III spectrophotometer (Pharmacia, Sydney, Australia). Total protein was calculated manually by reading the final absorbance from a standard curve constructed by measuring the same five aqueous protein standards as in the automated method. Diluted lyophilized sera (BioRad, Sydney, Australia) were used as controls in both methods. Samples with absorbances reading higher than the highest standard were diluted manually to fall within the standard curve. 2.3. Precision analysis

Intra-run precision was estimated by performing duplicate assays on a range of patient specimens and calculating standard deviation (S.D.) and coefficient of variation using the formulae [7]: S.D.=

/-CVX/ 2 N

S.D. -

X

d = difference between the duplicates in g/1 N = number of samples )( = mean of duplicates.

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Agreement was assessed graphically using Bland and Altman plots [8]. Inter-run precision was calculated using the same formula as above, but with duplicates performed in separate runs.

3. Results

3.1. Method comparison We compared the manual and automated methods by measuring 45 patient samples by both methods and found a correlation coefficient of 0.95. Least squares estimation of the closeness of fit showed a slope of 1.13 with an error of 0.004 and Y intercept of - 0 . 0 0 3 with an error of 0.006. The correlation line fitted the equation~ CB = 1 . 1 3 B C A - 0.004. The line of best fit equation indicates that the CB method reads higher than the BCA method. The Bland and Altman plot [8] in Fig. 1 shows that despite a high correlation coefficient of 0.95, there is poor agreement at higher protein concentrations.

3.2. Method precision Both within and between run precisions of the automated method were comparable to those of the manual BCA method (Table 1). Fig. 2 shows that the precision of the automated method was greatest at 1-1.5 g/1 protein. .-.

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Fig. 1. Difference between the manual BCA method and automated Coomassie Blue method plotted against the mean of protein quantitation. ~, mean of % differences; S.D., standard deviation of the mean differences.

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Table 1 Comparison of inter- and intra-run precision of the automated Coomassie Blue and manual B C A procedures Protein concentration

Method

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CV (%) - - within run - - between run within run between run

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83 27 41 38

For intra-run precision estimation, each sample was assayed in duplicate and their coefficients of variation (CV) calculated. F o r inter-run precision, each sample was measured with duplicates performed in separate runs, and coefficients of variation calculated. n, number of samples assayed.

3.3. Interference studies We examined the effects of various common buffers and detergents in samples of deionised water using the automated method, and confirmed that concentrated solutions of SDS, Triton X-100 and EDTA should be avoided when using this method (Table 2). Detergents and EDTA used at their usual concentrations, and Tris in a concentration of up to 2 mol/l, however, do not significantly affect the accuracy of protein quantitation.

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Fig. 2. Differences between duplicate Coomassie Blue measurements against the mean of the two duplicates, i, mean of % differences; S.D., standard deviation of the mean of % differences.

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A.J. Williams et al. / Clinica Chimica Acta 259 (1997) 129 136

Table 2 Effects of commonly used buffers and detergents on protein quantitation using the automated Coomassie Blue method. Each reagent was added to de-ionized water and apparent protein quantified. The results are the mean of 10 measurements Substance

Protein (g/l)

1% Sodium dodecyl sulfate (SDS) 0.1% SDS 1% Triton X-100 0.1% Triton X-100 1% Digitonin 0.1% Digitonin 2.5 mol/1 EDTA 0.25 mol/l EDTA 2 mol/1 Tris

0.36 0.02 0.57 0.02 0.08 0.02 0.38 0.03 0.08

4. Discussion

Traditionally, total protein in skin fibroblasts and other tissues is measured by a manual method, usually by the Lowry [1,2] or BCA [3] methods. We used a random access analyzer (the Roche MIRA) to measure protein by the Coomassie Blue method [6,9] and compared this to a manual BCA method. The Roche M I R A performed all of the processes required to analyze the amount of protein in the samples and even automatically diluted the samples when the absorbance was higher than that of the highest standard. The M I R A is a flexible random access analyzer and samples could be added or deleted and other tests performed on the same specimen during a run. Up to 60 samples could be loaded onto the MIRA at one time. We found that the automated method compared favourably to the manual method. There was no significant loss of precision in the automated method and it was significantly faster, taking 2 min per sample compared to 40 min by the manual method. Moreover, the precision of our method compares favourably with other published automated methods, including a microplate-based system [10], and automated flow injection analysis [11]. In our studies there were good intra- and inter-run correlations, but agreement was imperfect, especially at high protein levels when comparing the two methods (Fig. 1), and at low protein levels for the intra-run precision studies of our automated method (Fig. 2). Since the MIRA is capable of analyzing more than one sample at the same time, we were able to analyze 54 samples in 60 min. In addition, the automated method uses less sample, which is important where a number of

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biochemical analyses are to be performed on a limited a m o u n t o f sample. Moreover, sample with a protein concentration greater than 1.0 g/1 had to be manually diluted and reanalysed in a separate run using the manual BCA method, whereas the same sample was automatically diluted and reanalysed in the same run by the M I R A apparatus using the Coomassie Blue method. The Coomassie Blue reagent is inexpensive, readily available and stable for m a n y months at r o o m temperature. The automated Coomassie Blue m e t h o d costs 1 1 cents per test, while the BCA m e t h o d costs 35 cents per test. We found that the samples can be stored frozen for at least 6 months or at 4°C for at least a week with no loss of protein. For our studies we used three sequential steps for sample preparation to allow us to perform enzymatic assays, but sonication alone would suffice in preparing cell culture samples for protein quantitation using the automated method. The Coomassie Blue m e t h o d of measuring micro total protein is in c o m m o n use in m a n y routine clinical chemistry laboratories and although scientists working in research laboratories may not have immediate access to this equipment, the samples m a y be stored for up to 6 months at - 70°C and measured as a batch. We recommend that where the M I R A apparatus is available, consideration be given to its use for automated protein quantitation of cell or tissue extracts, because it is quicker, simpler, cheaper and as precise and reliable as manual methods.

Acknowledgements This research was financially supported by the Children's Hospital Fund, the Philip Bushell Foundation, and the Financial Markets Foundation for Children. We thank Dr Craig Mellis for his helpful statistical advice and for reviewing this manuscript.

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[6] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254. [7] O'Leary TD, Geary TD. Guidelines for the assessment of diagnostic systems for use in clinical biochemistry testing. Clin Biochem Rev 1990; 11:45- 80. [8] Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;(8476):307-310. [9] Heick HJM, Begin-Heick N, Acharya A, Mohammed A. Automated determination of urine and cerebrospinal fluid proteins with Coomassie brilliant blue and the Abbott ABA-100. Clin Biochem 1980;13:81-85. [10] Held PG, Absher M, Heintz NH, Hale PD. Automated procedures for the quantitation of protein. BioTechniques 1994;17:988-991. [11] Salerno RA, Odell C, Cyanovich N, Dubnis BP, Morges W, Gray A. Lowry protein determination by automated flow injection analysis for bovine serum albumin and hepatitis B surface antigen. Anal Biochem 1985;151:309-314.