Proteomics
Sample Preparation
Protein Sample Preparation
The Proteomics and Metabolomics Core offer following sample preparation services for biological samples such as cells, tissues and body fluids.
- Sample homogenization (e.g. Cells and tissues)
- Reduction, alkylation, digestion (in-solution or in-gel) and C18 stagetips cleanup
- Depletion of High Abundance Proteins (Agilent MARS-14) from serum/plasma
- Isobaric tagging for Relative and Absolute Quantitation (iTRAQ), TMT labeling for Protein ID & Quan
- Multidimensional protein and peptide seperation using UPLC
Please note:
- We only provide downstream LC-MS/MS analyses for SILAC labeled protein samples
- Based on the project requirements PMcore will decide to choose the sample prep services
Protein Digestion
In-gel digestions are most successful with Coomassie stained gels. In cases where higher sensitivity is required, silver stain or Sypro Ruby can also be used. When doing silver staining, make sure to use a mass-spec compatible staining kit. Silver stained gel spots should be submitted in 1% acetic acid. Coomassie or Sypro Ruby gel spots may be submitted dry (dehydrated) or in de-staining buffer (some sample loss may occur over time). Please refer to the sample submission page for specific instructions on how to process and submit your samples for analysis. Our standard In-gel and in-solution digestion protocols include reduction and alkylation of cysteine residues followed by enzymatic digestion with trypsin. Alternative enzyme digestions can be performed if needed, please contact us directly to discuss the available options and pricing. Following enzymatic digestion, samples are purified and concentrated using C18 tips or spin filters.
Please contact PM Core at pmcore.cpos@hku.hk or call 2831-5482 to consult which approach is compatible with for your research, and also for custom-made MS analyses for your compounds of interest. Pricing and scheduling will vary according to the complexity of analyses.
Protein Identification – ESI-LC-MS/MS
Workflow
In-solution/gel Digestion
Protein samples are digested with Trypsin/Lys-C or both.
LC-MS/MS Analysis
Protein Identification is performed on an on-line reverse-phase nanoLC coupled to an Orbitrap Fusion Lumos mass spectrometer.
Database search
The SEQUEST search engine is used to search the acquired MS/MS spectra, the resulting protein identifications are further validated using the Proteome Discoverer (PD) software.
Within the PD software, supplementary statistical analysis is applied to further validate the peptide identifications and assist in data interpretation process.
Results Delivery
The results will be delivered to users as a PD results file.
Please create an account at Proteome Discoverer Software website to download the 30-day trial version of the PD software.
With the viewer, you will have access to all of the raw MS/MS spectra acquired for your sample as well as a report of all of the parameters used in the data analysis for potential publication.
After 30 days the trial version will only act as a viewer of output files.
Discovery Proteomics
- Isobaric Tagging – Isobaric tagging is based upon chemical isotope incorporation with tags of identical chemical structure and the same total mass. The labile portion of the tag that is released during fragmentation (i.e. reporter ions) will vary in mass and relative quantitation is based on the intensity of these reporter ions. Isobaric tagging approaches enable multiplexing. iTRAQ, Tandem Mass Tag (TMT), ICAT.
- Metabolic Labeling – Stable isotope labeling using amino acids in cell culture (SILAC) involved incorporation of isotopes through the use of media containing 13C or 15N labeled amino acids. Relative quantitation is based on the intensities of the light and heavy labeled peptides. (Please note that we do not provide cell culture services but can support experimental design and proteomic analysis of SILAC labeled samples). SILAC.
- Label-free – Method aims to determine the relative amount of proteins in two or more biological samples. Unlike other methods for protein quantitation, label-free quantification does not use a stable isotope containing compound to chemically bind to and thus label the protein. Label-free quantification may be based on precursor signal intensity or on spectral counting. Spectral Counting is based upon the assumption that the number of MS/MS spectra identified for a given protein is directly proportional to the abundance of that protein in the sample. Spectral Counting can be used to obtain relative quantitation of proteins in a complex sample without the requirement of labeling
Please note:
- For these experiments we recommend triplicate analysis of each biological replicate, to increase statistical significance of the peptide (and thus protein) fold-changes measured. This type of experiment can be extended to studies including protein expression as a function of time given an external stimuli or gene knockout, for instance. Label-free differential expression is only available on separations utilizing a single dimension of LC prior to MS/MS
- Both Discovery and Targeted experiments are time and labour intensive. The success of these approaches relies on detailed planning and choosing the appropriate sample preparation techniques
Protein Identification
Untargeted Proteomics
Quantitative Phosphoproteomics
Metabolomics
Targeted Metabolomics
We provide MS-based targeted analysis for broad categories of metabolites (Detailed list: Download here).
- Fatty acids
- Amino acids and derivatives
- Tricarboxylic acids and Glycolysis intermediates
- Phosphate pentose pathway intermediates
- Bile acids
- Steroids
- Neurotransmitters
- Full service include consultation, sample preparation, GC or LC-MS/MS based relative or absolute quantitation, and data analysis.
- Sample preparation protocol will be decided based on sample type, for more details please go to our services -> sample requirements and platform applications.
- We also offer targeted metabolite quantitation for analytes from our in-house metabolite library (Fiehn’s metabolites) containing ~1400 compounds (Download here).
- The possibility of metabolites for quantitation outside the above in-house library can also be discussed separately with core facility scientists.
Please contact PM core at 2831-5486 or pmcore.cpos@hku.hk for project discussion and service quotation.
Targeted metabolite profiling workflow:
Full service package includes consultation, sample preparation, LC /and GC-MS/MS runs and data analysis
Guaranteed Service
Quality Control:
- We provide QC results upon request to demonstrate LC/GC & MS performance before project sample analysis.
- We run QC samples between project sample runs to assess overall system performance to ensure data quality suitable for publication purpose.
- We check criteria of standard calibration curves to ensure they are suitable for project sample quantitation.
- We check criteria of project samples to ensure relative or absolute quantitation and confirmation are valid.
| Injection type | Purpose | Criteria | Limit |
| Standard | Calibration | R2 | > 0.99 |
| Accuracy | +/- 30% | ||
| Project Sample | Quantitation | % CV (Technical replicate) | < 20% |
| Signal / Noise | > 10 | ||
| Confirmation | Retention time error against standard | < 0.2min | |
| Ion ratio error against standard | < 20% |
Criteria for standard calibration, sample metabolite quantitation and confirmation. More stringent limits are possible, subject to factors such as analyte concentration.
Service Charges
Click here for more details.
Sample Submission and Requirements
In order to ensure project success we offer consultation on project planning and experimental procedures best suitable to answer specific research questions using mass spectrometric methods on time-bound proteomics & metabolomics projects.
Please contact PM core at 2831-5484 or pmcore.cpos@hku.hk for a project consultation, customised analyses and sample submissions.
Sample submission for Proteomics services:
- Submit service request through iLab
- Please contact our colleagues in advance (via phone/email) before bringing the samples to the Core
- Fill in Proteomics Sample for ESI-LC-MS Submission Form (Orbitrap – Lumos and timsTOF), and attach it under service request in iLab
- Bring samples to the Core
Sample submission for Metabolomics services:
- Submit service request through iLab
- Please contact our colleagues in advance (via phone/email) before bringing the samples to the Core
- Fill in Metabolomics Sample Submission Form and attach it under service request in iLab
- Bring samples to the Core
Please refer to the FAQ section for frequently asked questions regarding sample submission and requirements.
The sample requirements for various applications on PM Core platforms are given below:
Platform Applications Sample type Sample preparation Platform requirement Orbitrap Fusion Lumos
(LC-MS/MS)
timsTOF Pro
(LC-MS/MS) – Protein ID
– Protein Quan (DDA or DIA)
– Phosphorylation site ID & Quan Tissue 2mg dry weight 50ng - 2µg peptides Serum / plasma 0.1 ml Cell lysate < 1 million cells or 5-20 µg protein.
5µg purified protein for Phospho site ID.
1mg – 2mg protein per condition for Phosphoproteomics GC-QQQ MS Targeted Metabolite Quantification Serum or plasma 0.2 ml per sample Metabolite concentration range:
pM – mM Tissue 20-50 mg per sample Cell lysate 3-6 million cells per sample LC-QTRAP and LC-OE120 MS Targeted Metabolite Quantification Serum or plasma 0.2 ml per sample Metabolite concentration range:
pM – mM Tissue 20-50 mg per sample Cell lysate 3-6 million cells per sample Kit based Targeted Metabolite Profiling Serum or plasma 10 µl per sample Cell lysate 3 million cells per sample Bio-HPLC Offline Fractionation (SCX, SAX, basic RPLC) Proteins isolated from any samples type n/a 500µg – 5 mg
Please note:
- Samples will not be accepted without prior consultation. Prioritization will be given to time-sensitive projects, including preliminary data needed for timely grant submissions and additional experiments needed for resubmission of manuscripts.
- We also receive ready to inject samples for LC-MS/MS analyses. However, prior to mass spectrometry analysis, samples should be salt, buffer, and detergent free. All other sample solution conditions must be discussed with the staff prior to sample submission. Providing the cleanest samples possible will greatly enhance our ability to provide you with the best mass spectrometry data.
- Turn around time: Typical 15 – 40 working days depending on project specifics/service basis.
Contact
Dr SHARMA, Rakesh
Dr LAM, Ronald
