Highly Efficient Extraction of Serum Peptides by Ordered Mesoporous Carbon

Abstract

Human serum is of great importance for organ function and the potential diagnosis of diseases. Serum biomarkers have been widely applied in clinical diagnosis and disease therapies. Endogenous serum peptides are an important class of potential biomarkers for the elucidation of biological and pathological variations in the serum. However, owing to the complexity and the highly dynamic range of protein concentrations in serum, the discovery of serum peptides, especially those at low abundance levels from minuscule blood samples, is still a challenge. With regard to obtaining serum peptides, organic solvent precipitation is a simple method for the removal of highly abundant proteins. However, the difficulty in discriminating between peptides and proteins together with the inevitable loss of peptides during the removal of proteins by solvent precipitation makes this method inefficient for the extraction of peptides. Centrifugal ultrafiltration (UF) with an accurate molecular weight cutoff is considered to be a very useful technology for the separation of proteins with low and high masses. By using UF coupled with a nanospray ionization hybrid ion trap/Fourier transform mass spectrometer, 300 unique peptides were identified from a 60 mL of serum sample. However, the coconcentration of small molecules and salts result in inefficient peptide extraction and severe interference to the MS detection. Therefore, additional peptide enrichment as well as salt removal by solid phase extraction (SPE), particularly using hydrophobic C18 adsorbents, has to be adopted before MS analysis. To simplify the extraction of peptides from serum, ordered mesoporous silica materials have been applied to enable the selective extraction of serum peptides rather than large proteins by the size-exclusion effect of the mesopores. Unfortunately, owing to the inherently insufficient hydrophobicity of silica some peptides are not extracted, thus resulting in a low number of unique peptides being identified. Surface modifications to mesoporous silica and titanium oxide materials have been developed for the enrichment of some specific posttranslational peptides, such as phosphopeptides and glycopeptides in serum. So far, a highly efficient general method for the extraction of a broad spectrum of serum peptides rather than for specific peptides is still absent; such a method would be a crucial technology for the discovery of serum biomarkers from very small blood samples. Herein, we describe the synthesis of an ordered mesoporous carbon material (OMC) and its use for the enrichment of a broad spectrum of endogenous peptides from serum. The expected high efficiency of this method for peptide enrichment is due to the distinct hydrophobicity of carbon as well as the size exclusion of the mesopores against serum proteins. The procedure for the extraction of a broad spectrum of endogenous serum peptides using OMC is illustrated in Scheme 1.