Isotope labeled internal standards (ILIS) as a basis for quality control in clinical studies using plasma samples Melinda Rezeli ⁎ , Ákos Végvári, György Marko-Varga, Thomas Laurell Clinical Protein Science & Imaging, Dept. of Measurement Technology and Industrial Electrical Engineering, Lund University, BMC C13, SE-221 84 Lund, Sweden ARTICLE INFO ABSTRACT Article history: Received 30 September 2009 Accepted 15 February 2010 For clinical proteomic studies, the quality of the biofluid samples such as human blood plasma is extremely important. In this study we have investigated the stability of human plasma samples by spiking stable isotope-labeled peptides into the plasma and monitoring their degradation under different storage conditions. FPA-1, C4A and C3f were synthesized with isotopically labeled amino acids, and used as reference peptides. The mixture of internal calibrants was spiked into plasma at the starting point of investigation, mimicking the time of collection for future biobanking efforts, and their qualitative and quantitative changes were analyzed over time by using both MALDI-MS (LTQ Orbitrap XL) and nanoLC- ESI-MS (LTQ XL ETD). We have found that all three synthetic peptides were stable in plasma at - 20 and - 80 °C during the examined 2-month period. However, different proteolytic degradation profiles of the peptides were observed at room temperature. We anticipate that the use of these isotope-labeled peptides as internal standards (ILIS) provides a quality control for long-term storage and proteomic plasma analysis. © 2010 Elsevier B.V. All rights reserved. Keywords: Clinical proteomics Heavy isotope-labeled peptides Human blood plasma Mass spectrometry 1. Introduction The human blood proteome has a great biological and clinical importance and reflects the environment of all cells, tissues and organs in the human body. However, the complexity and broad dynamic range of proteins make the characterization of blood, as a clinical sample type, a challenging task [1]. Due to the continuous development of proteomic techniques (mainly centered around mass spectrometric identification), the number of proteins annotated and identified in plasma/ serum in recent years has expanded to reach expression levels of many thousands of unique identities in biological samples [2,3]. However, there is still a demand for optimizing the process for characterizing clinically relevant proteins and to improve on the process of validating identity on as yet unidentified proteins within blood plasma. The combina- tion of high-abundant protein depletion and high-resolution liquid chromatographic separation interfaced to tandem mass spectrometry has enabled an in depth, and targeted approach where disease mechanisms and alterations have been ex- plored [4,5]. The majority of these proteins are medium or high abundant, but it is possible to identify proteins with concen- tration levels in the pg/mL region in human blood plasma [6]. Recently, the focus of proteomics studies has been transferred to quantitative aspects [7,8], where MRM-based assays permit a rapid and absolute quantification of numerous proteins in complex biological samples [9,10]. Profiling of human serum and plasma is of key importance, because these samples represent ideal compartments where biomarker discovery studies are undertaken. In this respect, blood sampling will provide a continuous reflection of healthy and disease status within the human body. Several biomar- kers and biomarker candidates related to cancer [7,11–14] and other diseases, such as myocardial infarction and diabetes JOURNAL OF PROTEOMICS 73 (2010) 1219 – 1229 ⁎ Corresponding author. Tel.: +46 46 222 3721; fax: +46 46 222 4527. E-mail address: melinda.rezeli@elmat.lth.se (M. Rezeli). 1874-3919/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2010.02.012 available at www.sciencedirect.com www.elsevier.com/locate/jprot