Magnetic mesoporous silica of loading copper metal ions for enrichment and LC-MS/MS analysis of salivary endogenous peptides
Graphical abstract
Cu2+ doped mesoporous silica-coated magnetic nanomaterial (designated as Fe3O4@mSiO2-Cu2+) was synthesized successfully and applied to enrich the endogenous peptides from human saliva.
Introduction
Cancer is the second leading cause of death around the world. Probably one out of four deaths is caused by cancer in developed countries [1]. In order to diagnose cancer at early stages before distant tissues are infected, a great number of methodologies have been used currently, among which peptidomics research of biofluids is a promising method for discovering potential biomarkers and monitoring human diseases [2].
In the past few years, mass spectrometry (MS) has become a conventional tool in studying endogenous peptidomics due to its fast speed, high efficiency and salt tolerance [3,4]. Up to now, researchers have found a lot of differences in endogenous peptidome between the samples from healthy and unhealthy people via mass spectrometry analysis, which have been proved to be closely related to specific disease [5]. As we all know, biofluid is a tremendous thesaurus for the discovery of peptide biomarkers [6]. It is considered that the endogenous peptides in human biofluid contain more information than the precursor protein as the protein always undergo proteolytic cleavage [1]. As a result, the analysis of endogenous peptides is believed one of the best ways for monitoring the physiological and pathological process of human being [7]. In recent years, saliva has aroused great interest among researchers for its noninvasive collection and easy availability as a traditional clinical sample [8]. In addition to low-abundance endogenous peptides, saliva comprises an array of bacteria, cellular debris, salts and proteins [9], all of which will interfere the identification of endogenous peptides due to the high dynamic range of bio-sample [10]. Besides, the microbial metabolites produced in the forms of soluble microbial products and extracellular polymeric substances during biological treatment which mainly consisted of proteins, carbohydrates, and humic substances. All of these interfered the analysis of peptidomics [11]. Therefore, it is urgent to develop an efficient method to remove those interference from complex saliva sample prior to MS analysis.
Mesoporous material is well known for its uniform porosity structure, high specific surface area and adjustable pore size [[12], [13], [14], [15]]. Up to now, several kinds of mesoporous materials, including mesoporous silica [13,16], mesoporous carbon [17] and mesoporous titania [18], have been applied in enrichment of peptides. The experimental results showed that the mesoporous material with pore size ranging from 2āÆnm to 5āÆnm possessed better enrichment performance towards peptides [19], which is able to exclude high molecular weight proteins while allowing small-sized peptides to enter into the mesopore based on size-exclusive mechanism [3]. Besides, mesoporous materials possess excellent property of chemical and thermal stability [20]. Therefore, researchers can modify the mesopore inwalls according to their actual demand. In that way, the interference brought by macromolecular proteins could be eliminated, which improved identification efficiency of endogenous peptides. In our previous work, L-Cysteine [3], C8 group [21] and so on have been selected to modify the mesopore inwalls for peptidomics researches. Consequently, all these work have achieved impressive results.
Immobilized metal ion affinity chromatography, denoted as IMAC, is one of the most effective techniques for peptidomics analysis. The mechanism of IMAC is believed as synergy of chelation [22] and electrostatic interaction [23]. Metal ions such as Cu2+, Ti4+, etc, immobilized on substrates can chelate with amino and carboxyl groups of the peptides through coordination bonds [24]. Subsequently, the peptides enriched on the ions can be released with an eluting reagent for the following MS analysis [25]. Copper ion as a transition metal ion shows an excellent property of chelation. No matter hydrophilic or hydrophobic peptide it is, copper ions exhibit a strong affinity to both of them [26]. So there is no doubt that copper ions play an important role in the field of IMAC for endogenous peptides analysis.
Herein, we proposed a combination of IMAC technique with mesopores and designed a Cu2+ doped mesoporous silica-coated magnetic nanomaterial (designated as Fe3O4@mSiO2-Cu2+) for highly specific enrichment towards endogenous peptides. After constructing a layer of mesoporous silica onto the surface of magnetic microspheres (Fe3O4), a thin adherent polydopamine film was covered upon the mesopore inwalls under an alkaline condition. With the help of polydopamine, the nanoparticles become more hydrophilic and biocompatible. Then, copper ions were introduced through chelation with hydroxyl groups of polydopamine. As we expected, the Fe3O4@mSiO2-Cu2+ showed a good performance in the enrichment of both standard peptides and complex clinical bio-samples.
Section snippets
Materials and chemicals
Iron (III) chloride hexahydrate (FeCl3Ā·6H2O), sodium acetate (NaAc), cupric sulphate (CuSO4Ā·5H2O), sodium hydroxide (NaOH), ammonium bicarbonate (NH4HCO3) tetraethyl orthosilicate (TEOS), ammonium hydroxide (NH3Ā·H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. Tris (hydroxymethyl) aminomethane was purchased from J&K Scientific. Cetyltrimethylammonium bromide (CTAB) was purchased from Adamas Reagent Co., Ltd. Dopamine hydrochloride was purchased from Alfa Aesar. Acetonitrile (ACN)
Characterization of Fe3O4@mSiO2-Cu2+
The fabrication approach of Fe3O4@mSiO2-Cu2+ nanocomposites was shown in Fig. 1. Briefly, Fe3O4 nanoparticles were prepared according to a hydrothermal method [27]. Then, a layer of mesoporous SiO2 was covered on the surface of Fe3O4 via solāgel process. With the removing of CTAB, a series of uniform silica mesopores were formed. Finally, Cu2+ was immobilized onto the mesopores through combination with a thin film of PDA covered on inwalls to obtain Fe3O4@mSiO2-Cu2+.
Some charactrization methods
Conclusions
In conclusion, a Cu2+ doped magnetic mesoporous silica material was proposed and applied to enrich the endogenous peptides from human saliva successfully. Thanks to the merits of mesopore and IMAC, Fe3O4@mSiO2-Cu2+ exhibited a great performance in identification of peptides from both standard protein digests and human saliva samples. Moreover, the good magnetic responsiveness made it easier for separation from mixture. As expected, the as prepared material showed a significant sensitivity of
Acknowledgments
This work was financially supported by National Key R&D Program of China (2018YFA0507501) and the National Natural Science Foundation of China (21425518 and 21675034).
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