Elsevier

Talanta

Volume 125, 1 July 2014, Pages 189-195
Talanta

Sequential depletion coupled to C18 sequential extraction as a rapid tool for human serum multiple profiling

https://doi.org/10.1016/j.talanta.2014.02.050Get rights and content

Highlights

  • A novel method for the differential proteomic analysis of human serum is presented.

  • The method relies in sequential chemical depletion for reducing serum׳s complexity.

  • Depleted serum is fractionated by sequential elution on C18 tips and analyzed by MS.

  • MS profiles are used for classification of samples from diseased and controls.

Abstract

Sequential chemical depletion of serum coupled to C18 sequential extraction of peptides as a rapid tool for human serum multiple profiling is herein presented. The methodology comprises depletion with DTT and then with ACN; the extract thus obtained is then summited to fast protein digestion using ultrasonic energy. The pool of peptides is subsequently concentrated using C18-based Zip-tips and the peptides are sequentially extracted using different concentrations of ACN. Each extract is mass-spectrometry profiled with MALDI. The different spectra thus obtained are then successfully used for classification purposes. A total of 40 people, comprising 20 healthy and 20 non-healthy donors, were successfully classified using this method, with an excellent q-value<0.05. The proposed method is cheap as it entails few chemicals, DTT and ACN, simple in terms of handling, and fast. In addition, the methodology is of broad application as it can be used for any study applied to serum samples or other complex biological fluids.

Introduction

Avoiding Serum׳s major protein components should be done by linking the protocol for depletion of such proteins to the proteomic profile of the disease under study [1], [2]. Following this reasoning, diverse chemical depletion methods have been developed and employed in proteomics. Depletion with ACN has showed to render a serum reach in apolipoproteins, adequate for studies dealing with cardiovascular-related diseases [1], [3], whereas, chemical depletion with DTT leads to a serum reach in immunoglobulin-like proteins, adequate to studies dealing with myelomas or lymphomas. In addition, DTT was found to be an excellent way to deplete major proteins whilst preserving an important number of them in solution [1], [4]. Very recently, the combination of a sequential chemical extraction using ACN and then DTT has been successfully applied to human serum for the searching of osteoarthritis biomarkers [5].

Also as a step prior to mass spectrometry analyses in proteomics, Zip-tips (or similar devices) are regularly used as a fast way to desalt samples as well as to pre-concentrate the peptides present herein [6], [7], [8], [9]. A step forward in the handling of Zip-tips for sample treatment in proteomics consists in the sequential elution of the peptides that are bound to the resin by using solutions of different composition [10]. Each solution extracts a different fraction of the peptides retained by the resin, which may be then employed for further research. This fractionation aids to reduce sample complexity, which ultimately leads to an increase in the number of peptides and proteins identified.

Finally, peptide profiling based on mass spectrometry has grown over the past 10 years to become the method of preference for the fast analysis of changes in protein expression patterns on biological systems, helping to achieve early disease detection, disease staging, therapeutic monitoring and prognosis of malignant diseases [11], [12], [13]. In the present work, the three steps mentioned above have been coupled for the first time (precipitations with DTT, with ACN and Zip-tips sequential elution), to the best of our knowledge, in order to develop an easy, fast and inexpensive method for the screening of human serum samples in proteomic studies aimed at biomarker discovery. We propose to deplete the samples with (i) chemical depletion promoted by DTT or (ii) by chemical sequential depletion. Then, to digest the remnant proteins, concentrate the correspondent peptides using Zip-tips and subsequently fractionate them by sequential elution with solutions of ACN of different concentrations. Finally, acquire MALDI-TOF mass spectrometry-based profiles of each eluate that can be analyzed with bioinformatics tools to find out differences between samples for classification purposes. The reproducibility and usefulness of this method are exemplified with the use of serum samples from a cohort of patients suffering from rheumatic diseases (RD) and healthy individuals.

Section snippets

Reagents

DL-dithiothreitol (DTT, ≥99%), iodoacetamide (IAA, ≥99%), trifluoroacetic acid (TFA, 99% FOR LC-MS), acetonitrile (ACN, LC-MS CHROMASOLV), water (LC-MS CHROMASOLV) and ammonium bicarbonate (≥99%) were purchased from Sigma (Steinheim, Germany) and were used for protein precipitation, reduction and alkylation. Trypsin (sequencing grade) from Roche (Mannheim, Germany) was used for protein digestion. NuTips large 10–200 μl and C-18 for peptide separation were purchased from Glygen (Columbia, USA).

Multiple profiling with DTT depletion

The sample treatment illustrated in Fig. 1 was followed in this set of experiments. The enzymatic digestion of a complex proteome generates a pool of peptides. Such peptides can be selectively separated through the interactions that they can establish with an immobilized solid phase, such as the one included in the C18-based NuTips©. C18 contains large hydrocarbon chains, which interact preferentially with hydrophobic peptides. As depicted in Fig. 1, in this optimization study we used a C18

Conclusions

Sequential chemical depletion of serum coupled to C18 sequential extraction of peptides as a rapid tool for human serum peptide profiling has been successfully proven. The methodology comprises depletion with DTT and then with ACN, and the extract thus obtained is then submitted to fast protein digestion using ultrasonic energy. Next, the pool of peptides obtained is concentrated using C18-based Zip-tips and the peptides are sequentially extracted using different concentrations of ACN. Each

Acknowledgments

C. Ruiz-Romero is the recipient of a Miguel Servet Grant (CP09/00114) from ISCIII-Spain. The Scientific Society ProteoMass is acknowledged for providing financial support. This work was supported in part through funding from the Fondo Investigación Sanitaria, Madrid, Spain (CIBER- CB06/01/0040; PI12/00329; PI 11/02397; RETIC-RIER-RD12/0009/0018; and Proteo-Red/ISCIII); Ministerio Ciencia e Innovación (PLE2009-0144), FEDER (European Community) and Xunta de Galicia: Grant 10 PXIB 310153 PR and

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