1. For shot-gun sequencing method, please list at least THREE factors that affect the measurement of proteins in different samples?
- Characteristic Ion Selection
- Instrument performance: Resolution & Mass accuracy
- Sample preparation
2. What is SILAC and why in most SILAC experiments, “heavy” arginine and lysine are used in the cell culture?
SILAC is an acronym for “Stable Isotope Labeling with Amino acids in Cell Culture”
They are the targets of trypsin. When proteins are then digested with trypsin, each protein has a single “heavy” amino acid resulting in a consistent mass shift.
3. Considering the simple procedure with protease digestion, please state the reason why protease digestion has not been used as common as we expected.
- The difference in mass between light and heavy peptides is small, making quantitative analysis more complicated
- In low resolution MS and electrospray ionization MS, the exchange (from H218O to carboxyl group) may be blocked by urea
- Back-Exchange & Structural-specific exchange rate
4. For peptide HTSGPPFKTNR, after demethylation with D13CDO and NaBH3CN, what is the mass difference between heavy and light peptides?
5. Can you make a table to compare at least four major difference between SILAC and iTRAQ labeling methods?
|Labeling Method||in vivo||in vitro|
|Suitable sample||passageable cells or bacteria||no limit|
|Quantitative analysis||First-level mass spectrometry||Second-level mass spectrometry|
6. There is a newest paper that reported the new TMT reagents called TMTpro. What is new with the TMTpro reagent?
TMTpro label reagents are the next generation of tandem mass tags, designed to increase the level of sample multiplexing without compromising on protein identification and quantitation. The TMTpro tag structure is similar to the TMT tag structure in that the tags remain isobaric and amine reactive, but differs in the longer spacer region and isobutyl proline mass reporter region. After MS/MS fragmentation, each TMTpro tag generates a unique reporter mass (i.e., TMTpro 126-134Da) in the low-mass region of the high-resolution MS/MS spectrum that is used for relative quantitation of protein expression levels.
The advantages of TMTpro tags include increased multiplex relative quantitation, increased sample throughput, and fewer missing quantitative values among samples. The TMTpro 16plex label reagents are suited for analyses of multiple protein samples, such as inhibitor dose response experiments, time course experiments, thermal shift assays, or biological replicates. When combined with the industry-leading, high-resolution Orbitrap instruments and software, TMTpro reagents provide integrated total solutions for quantitative protein expression analysis.
7. What is the AQUA method and please briefly describe the method.
Absolute Quantification is a targeted quantitative proteomics technique that exhibits robust efficacy and is being increasingly utilized for a wide variety of quantitative proteomics studies. AQUA strategy is for the absolute quantification (AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e. g. , phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and post-translationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer.
8. List at least three methods to improve label-free quantitation.
- Improve the resolution, stability and sampling speed of liquid chromatograph and mass spectrometer
- Improve the efficiency of quantitative algorithm
- Enough technical repetition