Sequential depletion coupled to C18 sequential extraction as a rapid tool for human serum multiple profiling
Graphical abstract
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
References (15)
- et al.
Anal. Biochem.
(2009) - et al.
J. Pharm. Biomed. Anal.
(2008) - et al.
J. Chromatogr. A
(1998) - et al.
Anal. Chim. Acta
(2011) - et al.
J. Proteomics
(2012) - et al.
Electrophoresis
(2011) - et al.
Electrophoresis
(2010)
Cited by (7)
Ultrasonic-assisted extraction and digestion of proteins from solid biopsies followed by peptide sequential extraction hyphenated to MALDI-based profiling holds the promise of distinguishing renal oncocytoma from chromophobe renal cell carcinoma
2020, TalantaCitation Excerpt :Among these extraction procedures, on-a-tip SPE strategies has been widely applied in the proteomic field, specially C18 resin packed on-a-tip. Thus, the sequential extraction of peptides with C18-based tips has been used to reduce proteome complexity of biological samples through sequential extraction of peptides followed by mass spectrometry interrogation with the aim of finding biomarkers of diseases [11]. Taking into consideration the problems and solutions described above, a methodology to elucidate differences between chromophobe renal cell carcinoma and renal oncocytoma has been developed as follows.
Simplifying the human serum proteome for discriminating patients with bipolar disorder of other psychiatry conditions
2017, Clinical BiochemistryCitation Excerpt :The 1-DE profile obtained after the sequential depletion (see Fig. 1, panel B) showed a decrease on high molecular weight proteins that is characteristic of ACN-depleted serum, and also the enrichment of some proteins that were masked by albumin in the crude sample, thus characterizing the DDT-depletion. The results obtained with this combined depletion method, support the data found by DTT- and ACN-depletion previously reported above [21–23,27]. Finally, the results regarding PM are displayed in Fig. 1 (panel B), in which we observe a lower gel band density, confirming the decrease on dynamic range of proteins in the sample, including albumin (~ 70 kDa), immunoglobulin (~ 25 and 55 kDa) and transferrin (~ 79 kDa).
A cost-effective method to get insight into the peritoneal dialysate effluent proteome
2016, Journal of ProteomicsCitation Excerpt :Furthermore, the proteins retrieved from different depletion methods are not the same since the type, and characteristics of the proteins depleted are different [7]. Recently, acetonitrile, ACN, and dithiothreitol, DTT, have been reported as simple, reproducible and cost-effective methods to remove high-abundance proteins from complex matrices, such as serum [7–9]. Indeed, serum protein depletion with acetonitrile or protein equalization with DTT has proven to be convenient to enrich selectively serum extracts in apolipoproteins and immunoglobulins, respectively.
Prognostic model to predict the incidence of radiographic knee osteoarthritis
2024, Annals of the Rheumatic DiseasesImplementing effective machine learning-based workflows for the analysis of mass spectrometry data
2016, Journal of Integrated OMICS
- 1
URL: www.bioscopegroup.org.