Through pH modulation of the peptide sample, a peptide’s interaction with the nanoporous layer of the nHance™ PCS – Peptide Capture System can be manipulated to provide superior peptide extraction.
Peptide Recovery: pH Modulation
Bioanalytical identification and quantification of specific molecular components in body fluids is a critical enabler of clinical translation of both drugs and diagnostics. Two independent trends in clinical treatment have brought challenges in assay development and deployment. In drug discovery, small molecule drugs have given ground to biologic drugs, with a natural first step in moving up the molecular weight scale opening the door to peptide therapeutics. In diagnostics, circulating biological molecules are now seen as communication vectors – biomarkers, signaling the presence of disease states even at their earliest onset. The regime of circulating biomolecules in the range [1kDa ~ 10 kDa], which generally includes peptides, represents a rich regime in which biomarkers are being sought and found (see Figure. 1).
A translational challenge for the development of both drugs and diagnostics is developing and deploying assays for identifying and quantifying biomolecules in the peptide regime. The gold standard for biomolecule analysis has traditionally been ELISA. ELISA assays can be specific and sensitive; however, they can be expensive to run due to reagent costs and typically are not able to distinguish isotopes or monitor catabolism or post‐translational changes of peptides and proteins in vivo. Furthermore, in drug discovery, where speed matters, for a new target biomolecule it may take months to culture the necessary antibodies for an ELISA assay.
Liquid Chromatography Mass Spectrometry (LC‐MS) has emerged as a powerful technique in modern assays. LC‐ MS instruments can separate and quantify biomolecules based on affinity properties (e.g., hydrophilicity/hydrophobicity) as well as precision measurement of molecular weight. Exponential improvements in computational technology have advanced the technical design control and real‐time data analysis capability of the mass spectrum instruments.
To deliver high‐quality quantification results, an LC‐MS instrument requires sample preparation techniques specifically suited to the manipulation of peptides and proteins. Solid‐Phase Extraction (SPE) was originally well characterized for small (non‐biologic) molecules as a sample preparation technique for LC‐MS; however, the transition from small molecule to biologic molecule analysis has had mixed results. For example, SPE assays are not easily developed for macromolecules which tend to stick to the solid phase sorbent rather than elute as a bolus during the elution step of the protocol. To overcome this problem, scientists must incorporate other techniques (e.g. ELISA) to capture the large molecules. These combination assays can be extremely complex, requiring long incubations times, extensive wash and digestion steps, and sample preparation work flows at least 4‐6 hours post incubation, resulting in high total cost per sample. Therefore, there is a great industry need for a simple, straightforward, and cost effective sample preparation technique for peptide and protein analysis by LC‐MS.
Then nHance™ PCS – Peptide Capture System technology features an innovative proprietary material that is designed to enhance the detection of low abundant, low molecular weight peptides present in biological fluids such as urine, serum, CSF, and cell lysates. Peptide biomarkers are a measurable substance in an organism whose presence is indicative of some phenomenon such as disease, infection, or environmental exposure. Peptide quantification is also critical for drug development to understand drug metabolism and activity.
ThennHance™ PCS – Peptide Capture System technology employs a nanoporous silica material that is specifically engineered to sequester such low abundancy analytes, such as peptides, from biological fluids. “Nanoporous” describes a material that features a pore network, in this case a “sponge‐like” configuration, with pore diameters of approximately 4‐6 nanometers.
When biological fluid samples are introduced into the nHanceTM material, peptides and small proteins are able to enter the pore network of the nanoporous material, however larger proteins, such as albumin, are restricted due to their physical size. A washing step is used to effectively remove these highly abundant large proteins from the sample. The captured biomarkers, or low abundant peptides and small proteins, are then extracted from the nanopores by using a chemical solution tailored to release (elute) the specific peptides trapped in the pores. The solution containing the analytes of interest can then be introduced into a bioanalytical tool for subsequent analysis and quantification.
Sample Treatment: pH Modulation
The nHance™ PCS – Peptide Capture System 96‐well plate is a novel platform for the separation and enhancement of peptides. The proprietary thin‐film at the bottom of each well features a nanoporous silica layer with a negatively charged surface. The two critical parameters that impact peptide recovery when using this next generation product, include:
- The size of the peptide or protein in relation to the average pore size of the nanoporous layer; and
- The complimentary electrostatic interaction between the nanoporous layer surface and peptides of interest.
The first of these parameters is relatively straightforward; the pore size must be able to accommodate the volume of the target analyte of interest. The second parameter is more complex since peptides can feature both acidic and basic regions (zwitterionic peptides), so peptidomic responses to pH adjustments are difficult to predict. Table 1 below provides general guidance on recommended pH adjustments to a sample solution for peptides with various isoelectric points (pI). For example, if a peptide of interest features a pI of 5, it is recommended that the pH be adjusted to approximately 4. This provides a complementary, positive (Dark Orange), to slightly positive (Light Orange), peptide charge state that can more favorably interact with the neutral (Grey) to slightly negative(Light Purple) nHance™ nanoporous surface. Achieving complimentary ionic states between the peptide and the nHance™ surface facilitates peptide capture within the nanoporous material.
To confirm that a peptide’s interaction with the nanoporous layer of the nHance PCS can be manipulated through the alteration of the sample’s pH, a mixture of peptides and proteins ranging in size, pI, and hydrophobicity/hydrophilicity was prepared in non‐biological matrix (see Table 2).
The mix consists of acidic to basic (positively to negatively charged) molecules ranging in size from approximately 750 Daltons to 66,000 Daltons. It is hypothesized that by altering the pH of the sample prior to loading it into the nHance PCS, the net charge of the peptides and proteins will change. As the pH gets lower than a whole pH unit below the pI of the molecule, the net charge of the peptide becomes closer to neutral or positive increasing the molecules ability to interact with the nanoporous surface. Larger proteins will not be able to enter the nanoporous layer pores and therefore will not be retained on the device.
For the experiment, equal aliquots of the peptide mixture in neat solution were taken and adjusted (titrated) to different pHs ranging from 1.5 to 12. Before analysis, the relative retention of each peptide was predicted based on its size and its individual pI (Table 2). These predicted retentions were divided into three categories: detected, trace, and not detected.
Each pH adjusted sample was analyzed via mass spectrometry and the observed peptide measurements were also plotted using the same categorization. The observed responses, as predicted by the individual peptide’s pI and size, matched the theoretically predicted responses as shown in Table 3.
A total of 48 scenarios were tested and 95% of the scenarios (46 of 48) matched or exceeded the predicted performance. The nHanceTM PCS technology is the first product specifically engineered to address the clear and present need for a peptide enrichment tool for the expanding biologics market. To meet this challenge, the nHanceTM PCS technology was designed using state‐of‐the‐art semiconductor fabrication methods to create a peptide capture mechanism predicated upon size exclusion, provided by the pore dimensions of the nanoporous silica material, and the electrostatic interaction of the nHanceTM surface and target analytes.
The nHance™ PCS Advantage
The nHance™ PCS technology provides users unprecedented flexibility to employ one product to meet their bioanalytical needs for a range of target peptides.
- Significantly reduces method development time;
- Avoids complex, time consuming, and expensive sample pre-treatment steps;
- Does not require proprietary supporting equipment (e.g. vacuum manifolds); and
- Provides maximum interaction time between capture surface and target analytes that increases peptide capture efficiency compared to SPE and other “flow-through” enrichment devices.
Our application team includes experienced bioanalytical chemists with a significant knowledge base in the use of LC/MS techniques for the detection and quantification of peptides and proteins. Contact us today to see how the nHance™ PCS can simply your peptide and protein sample preparation for LC/MS analysis.