Determination of peptides and proteins in human urine with capillary electrophoresis–mass spectrometry, a suitable tool for the establishment of new diagnostic markers

doi:10.1016/S0021-9673(03)00713-1

Journal of Chromatography A

Volume 1013, Issues 1–2, 26 September 2003, Pages 173-181

https://doi.org/10.1016/S0021-9673(03)00713-1 Get rights and content

Abstract

The on-line coupling of capillary electrophoresis (CE) with electrospray-time-of-flight mass spectrometry (MS) has been used to obtain patterns of peptides and proteins present in the urine of healthy human individuals. This led to the establishment of a “normal urine polypeptide pattern”, consisting of 247 polypeptides, each of which was found in more than 50% of healthy individuals. Applying CE–MS to the analysis of urine of patients with kidney disease revealed differences in polypeptide pattern. Twenty-seven polypeptides were exclusively found in samples of patients. Another 13, present in controls, were missing. These data indicate that CE–MS can be applied as powerful tool in clinical diagnostics.

Introduction

Proteomics gains increasing importance as a new tool for identification of therapeutic targets [1], [2]. One of the ultimate challenges is to gain insight into the proteins found in body fluids such as urine and blood. This information could subsequently be used to define the state of health of an individual [3]. We set out to combine the high-resolution properties of capillary electrophoresis (CE) with the powerful identification ability of mass spectrometry (MS). CE is known as an efficient and rapid separation technique with high resolution, which is frequently employed in the separation of biological macromolecules such as proteins [4], peptides [5] and nucleic acids [6]. MS is currently one of the most powerful techniques to analyze biological samples, allowing identification based on the accurate mass of the molecules [7], [8], [9], [10], [11], [12], [13]. Therefore, the on-line coupling of CE and MS offers an attractive alternative to common chromatographic separation techniques. The advantages of CE–MS are speed, high resolution, sensitivity, and reproducibility [14]. To date, CE is applied to the separation of proteins [15], [16], but the combination of CE and MS to analyze polypeptide patterns in human body fluids is not yet established. To date, only a few single proteins or peptides were investigated with CE–MS [17], [18], [19], [20]. We have developed an on-line combination of CE–MS to depict polypeptides found in body fluids under the assumption that this will lead to the establishment of polypeptide patterns typical for the state of health of individuals.

Urine analysis provides a fast, non-invasive diagnostic tool for patients with renal diseases. Healthy individuals excrete less than 150 mg/day of protein in urine, while the urinary protein excretion of patients with renal diseases may exceed several grams a day [21]. Thus, the evaluation of these proteins may lead to an increased understanding of renal physiology and possibly allows the differentiation of subgroups of renal diseases by the identification of these proteins via CE–MS. Such data would eventually even make renal biopsy superfluous.

Here we show that the simultaneous assessment of a large number of proteins and peptides in urine is possible and leads to different, distinct polypeptide patterns in renal diseases.

Section snippets

Sample preparation

Best reproducibility of results without much degradation was found when spontaneous urine samples were analyzed repeatedly. Spontaneous urine samples of patients and healthy volunteers were obtained after informed consent and stored at −20 °C until further analysis. Urine samples were thawed and 2 ml was applied onto a Pharmacia C₂ column (Amersham Biosciences, Buckinghamshire, UK) to remove urea, electrolytes, salts and other interfering components, to decrease matrix effects and to enrich the

CE–MS conditions

As described in more detail by Kaiser et al. [22], we have developed a stable on-line combination of CE–MS to analyze the polypeptides present in body fluids. As shown in Fig. 1, more than 1000 polypeptides can be detected in the urine of a healthy individual with this approach. The overall pattern and the mass/charge ratios observed by repeated screening of the same sample as well as in comparison to other individuals of the same group were highly reproducible. The reproducibility of the

Acknowledgements

We are grateful to Jürgen Maier (Applied Biosystems) for excellent technical assistance. This work was supported in part by grant No. 0312939 from BioProfil “Funktionelle Genomanalyse”.

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Determination of peptides and proteins in human urine with capillary electrophoresis–mass spectrometry, a suitable tool for the establishment of new diagnostic markers

Abstract

Introduction

Section snippets

Sample preparation

CE–MS conditions

Acknowledgements

J. Biol. Chem.

Curr. Opin. Biotechnol.

J. Am. Soc. Mass Spectrom.

J. Chromatogr.

J. Am. Soc. Mass Spectrom.

Biochem. Biophys. Res. Commun.

J. Am. Soc. Mass Spectrom.

Biomol. Eng.

Clin. Chim. Acta

Lancet

Anal. Chem.

Angew. Chem., Int. Ed. Engl.

Dis. Markers

Electrophoresis

Electrophoresis

Electrophoresis

Electrophoresis

Chem. Rev.

Science

J. Chromatogr. B