1. 1D and 2D Gel Electrophoresis
1D and 2D electrophoresis are classical protein analysis methods for separating and visualizing protein mixtures. Combining this analysis with mass spectrometric protein identification makes it a very powerful tool for charcacterization of protein mixtures.
1.1. 1D SDS PAGE is used to look at less complex samples and to see the most abundant proteins. It is also very useful for comparing different production batches on a single gel. Silver staining makes it very sensitive and can be combined with our protein identification analysis.
1.2. 2D PAGE separates soluble proteins with isoelectric points from pI 3-10 and Mw from 10-130 kDa. Protein spots can be cut out from the gel and identified by a mass spectrometric analysis and database search, providing the protein name and database accession number. Typically 200-500 protein spots can be observed and identified in a silver stained 2D gel. The images obtained after 1D/2D gel analysis are scanned with a densitometric scanner providing semi-quantitative numbers for individual bands or spots.
1.3. 2D-DIGE is a gel-based approach for comparative proteomics using fluorescent tags. Distinct fluorescent tags e.g. Cy 3, 5 and 2 are used to label samples and a universal internal standard prior to 1st/2nd dimension electrophoresis. An automated software program is used to detect, quantify and annotate differentially expressed proteins. 2D-DIGE offers all the advantages of 2D-PAGE and overcomes the inherent disadvantage of variation and reproducibility problem in a 2D-PAGE.
2. Protein identification by linear-ion trap mass spectrometry
Digested protein samples will be then loaded on to a peptide trap at the front-end of an ion trap mass spectrometry (LTQ, Thermo Scientific) by a HPLC auto-sampler and washed by a HPLC pump to remove contaminants. A 40-minute acetonitrile gradient will then be applied to the peptide trap by another HPLC pump to elute the peptides sequentially onto a C18 reverse column for further refined separation. Separated peptide will be introduced into the MS via ESI interface, followed by automated MS and MS2 scans to collect the information for parent and fragment ions. Collected MS and MS2 spectral will be submitted to protein database search and protein identification results will be then generated for each protein samples.
3. Comparative proteomics using spectral-counting or TIC intensity methods
Depending on the complexity of the protein sample, a 1D HPLC separation (reverse phase) or a 2D HPLC separation scheme including strong cation exchange (SCX) and reverse phase (RP) will be used for separation and increase the proteome coverage. Separated peptide will be introduced into the MS via ESI interface, followed by automated MS and MS2 scans to collect the information for parent and fragment ions. Collected MS and MS2 spectral will be submitted to protein database search and protein identification results with 1% false discovery rate (FDR) will be then generated for each protein sample. All the protein identification results will next be imported into a proteomic quantification software to generate the protein abundance information based on the number of MS2 spectral or total ion chromatograph (TIC) intensities, which will be then be integrated into one excel file containing both protein identifications and quantification information.
4. Protein/peptide/small molecules quantification using SRM/MRM MS
Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) MS analysis of targeted molecules will be performed on a 4000 QTRAP hybrid MS from ABSciex. Both peptides and metabolites can be analyzed and quantified using SRM/MRM. One or multiple transitions will be needed for each target molecules, together with MS parameters, including collision energy (CE), de-clustering potential (DP) and etc. The method development by PCL for SRM/MRM experiments, i.e. transition identification and optimization and HPLC optimization are strongly suggested for each customer since it is highly instrument dependent. After MS analysis, SRM/MRM spectral will be processed using Multiquant software to extract peak areas for all transitions, which will then be exported to excel file and handed to customer. Further in-depth multi-variant analysis is also available upon request and at additional charge.
5. Protein quantification using high throughput ELISA/LUMINEX assays
Both ELISA and LUMINEX are performed in 384-well format (96-well format also available but less preferable due to increased plate-to-plate variations and large reagents consumption). For ELISA assay, both sandwich- and competitive- type of assays can be performed. LUMINEX assay is done on a FLEXMAP 3D system (Luminex Corporation, Austin, TX, USA), which is capable of measuring up to 500 proteins from a small sample volume. Assay kits can be either provided by the customers or purchased from the core facility at a discount price.
We provide metabolomic services for different types of samples, including tissue, serum, plasma, urine, etc. Sample extraction can be done by PCL at additional charge. Metabolites separation can be currently be done either using reverse phase column (for non-polar compounds) or HILIC column (for compounds) or both. Other HPLC separation can also be performed if the customer provides us with the column and protocols. Metabolomic profiling study (untargeted, discovery type of study) and targeted metabolomic study will currently both be performed on the 4000 QTRAP hybrid MS from ABSciex. The former will be done using Q1 MS scan to generate MS spectral, which will then be processed by Markerview software to extract peaks and elution time. An excel table with all the m/z, extracted peak area and elution time for all samples will be generated and given to customer. Further in-depth multi-variant analysis is also available upon request and at additional charge. Targeted metabolomic study will be described in SRM/MRM section.
High performance liquid chromatography (HPLC) separation and diode-array detection services on an Agilent 1200 HPLC are provided for a variety of molecules, including proteins, peptides, metabolites and other small molecules. We have reverse phase columns (C4, C8 and C18) readily available in our facility. Other columns will need to be provided by customers.
8. Sample preparation
8.1. In-gel digestion
Protein bands in PAGE gel need to be cut by the customer and transferred into 1.5-ml eppendorf tubes with molecular biology grade water just enough to cover the gel slices. The tubes will be submitted to Proteomics core lab (PCL) together with a sample submission form. The gel slices will then be pre-processed before digestion, including disulfide reduction with DTT and sulfhydryl alkylation with iodoacetamide. An overnight trypsin digestion for the gel slices will be then performed. Digested peptides will be extracted and concentrated the next morning after digestion.
8.2. In-solution digestion
Proteins in compatible buffer solution (PBS, Tris-HCl, water, etc) will be first tested for concentration if not known. A protein concentration step will be considered if the protein content is too low. A buffer exchange using gel filtration spin column will then be performed to change the buffer to 50mM ammonia bicarbonate. The protein samples will then be pre-processed before digestion, including disulfide reduction with DTT and sulfhydryl alkylation with iodoacetamide. An overnight trypsin digestion for the gel slices will be then performed. Digested peptides will be cleaned up if necessary and concentrated the next morning after digestion.
8.3. Protein/peptide fractionation using liquid phase IEF
Protein/peptide samples can be fractionated based on their isoelectric points using the Agilent 3100 OFFGEL Fractionator. The resulting fractions are in solution, making recovery for LC/MS analysis much easier than with conventional electrophoresis or 2D gels and suitable for mass spectrometry analysis following proteolytic digestion directly. The OFFGEL is also suitable for preparative peptide isoelectric focusing.