David Sarracino (Presenter)
Thermo Fisher Scientific BRIMS
Authorship: David Sarracino (1), Scott Peterman (1), Mazi Mohiuddin (1), Scott Kronewitter (1), Shen Luan (1), Amol Prakash (2)
(1) Thermo Fisher Scientific BRIMS, Cambridge, MA (2) Optys Technologies, Inc. Boston, MA
| Short Abstract Peptidomic profiling offers an alternative approach to identifying native peptide biomarkers in various fluids. The peptidome contains analytical targets preserving the endogenous formation such as signaling peptides and can be used to identify the specific proteolytic cleavage sites or unique PTMs. We present a comprehensive sample preparation, data acquisition, and analysis for studying the urine and plasma peptidome. Experiments were performed using bottom-up sample preparation and analysis to build the sub-proteome used to process the top-down data. Evaluation of the depth and reproducibility of the method is presented as compared to standard data processing approaches. |
Long Abstract
Introduction:
Peptidomics provides significant insights into biological activities through profiling “native” peptides. Peptidomics preserves the endogenous information such as proteolytic activity and PTMs and studies focus on identifying putative biomarker panels. The challenge with peptidomics is primarily attributed to sample preparation, detection, and data analysis. Typically, sample preparation is performed using solid phase extraction (SPE) and/or cation/anion exchange (CX/AX) to concentrate, clean, and extract the desired molecular weight (MW) range for automated data analysis and processing.
The primary challenge in performing peptidomics is balancing the extraction process to separate the peptidome (<30,000 amu) from the rest of the proteins in the sample. The extraction step must be reproducible, scalable, and automated. We have utilized a cation exchange resin to combine sample preparation steps to extract the desired peptidome MW range. The resulting samples could then be rapidly screened using a size exclusion chromatography (SEC) for high resolution/accurate mass (HR/AM) MS and MS/MS analysis to perform qualitative and quantitative peptidomic analysis.
The robustness and reproducibility of the method was evaluated using a standard urine and plasma sample. Success of the method was evaluated based on feature detection as a function of loading capacity and sample complexity across technical replicates.
Methods:
A stock urine sample was collected and pooled from a healthy donor over a 12-hour period a final volume ca. 1 L. The samples were then acidified to 2% with acetic acid and stored at 4oC until use. Plasma was collected from the same donor using tubes with protease inhibitors and stored until use. Samples were created by loading different volumes from the urine and plasma stock solutions. Urine was loaded from 500 to 12,000 µL and plasma was loaded from 25 to 1000 µL into separate SOLA Retain CX 10 mg wells. The samples were successively washed with 400 µL of water and organic prior to extracting the peptidome using 400 µL of an ammonium formate salt solution. The plate with the extracted peptidome samples was lyophilized and reconstituted in 40 µL of a 95:5 solution of A) 0.2% formic acid and B) MeCN (0.2% formic acid), which also was used as the binary solvents for chromatographic separations. Each level was prepared with 6 replicates to determine robustness and reproducibility.
All LC-MS data was acquired using normal flow rates (200 µL/min) with the binary solvents described above. Top-down data was evaluated using a rapid SEC separation and HR/AM MS and MS/MS analysis using a Q Exactive High Field quadrupole Orbitrap mass spectrometer. The SEC column dimension was 7.8 x 150 mm with in-house packing material and separation performed using an isocratic gradient of 18% B for 30 minutes. The bottom-up sample analysis was performed by first digesting the extracted peptidomic samples using standard tryptic digestion protocols, loaded onto a 2.1 x 250 mm column. Separations was performed using a linear gradient for 30 minutes.
Bottom-up peptidomics data processing was done using Proteome Discoverer 2.1 and searched against the human Uniprot database to build the protein list used to search the top-down data, which was processed in the Pinnacle software. Reproducibility analysis was analyzed in Pinnacle software to evaluate overall scoring in method performance.
Results
The goal of the study is to develop a comprehensive and robust analytical workflow for peptidomic profiling of biological samples. The method presented facilitates automated sample preparation with top-down and bottom-up data analysis and processing based on the desired sample analysis steps. Acquisition using the Q Exactive HF mass spectrometer providing significant resolution (>120,000 for MS and >60,000 for MS/MS) to facilitate qualitative and quantitative processing based on precursor charge state and isotopic distribution profiles and product ion series confirming sites of truncation. For the C18 separation, accurate retention time acceptance criteria of +/-1% was used for further confirmation at the MS and MS/MS events.
The initial experiments were performed using bottom-up data analysis to identify putative proteins from the Uniprot database in a predictable manner. Standard data dependent acquisition methods facilitated tryptic peptide identification. The proteins associated to the identified peptides were incorporated into a sub-proteome database used to process the top-down data. The initial urine peptidomic analysis identified ca. 450 peptides from a healthy donor. The peptides were routinely measured across all loading volumes with %CVs less than 25% for the SEC analysis and 20% using the C18 column.
The initial analysis of the plasma peptidome identified ca. 1250 peptide measured across all loading amounts. Similar to the urine study, the bottom-up analysis was used to create the protein database used to search the top-down data for intact and truncated forms of the peptides. The reproducibility measurements were similar in plasma as that measured in the urine peptidome despite the increased complexity.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |