Chem 250 Unit 1 Proteomics by Mass Spectrometry

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1 Chem 250 Unit 1 Proteomics by Mass Spectrometry Article #1 Quantitative MS for proteomics: teaching a new dog old tricks. MacCoss MJ, Matthews DE., Anal Chem Aug 1;77(15):294A-302A. 1. Synopsis 1.1. This article aims to describe some of the ways that isotope dilution is used to add quantitative accuracy to the MS of proteins in complex biological mixtures. It s a little bit difficult to analyze in depth because it touches on so many different aspects of a very extensive field Biochemistry and the analysis of proteins (complex macromolecules) is one aspect, electrophoresis, ion exchange chromatography and reverse-phase chromatography are another, sample introduction for MS, mass analyzers in MS and MS-MS (e.g. product ion scanning) is another Stable isotope labeling strategies are reviewed and fall into three main categories: digestion then labeling, labeling of entire proteins (probably followed by digestion), and adding labels to cell culture to label everything If you digest and then label, you get a complex, but highly unique fingerprint If you label at the protein level, you get a selective measurement by using an enzyme can be used for the labeling and that will make it label only specific proteins in a mixture (for example, those with peripheral cysteines). Selectivity also comes from the mass, but this is less distinctive than in a tryptic digest that gives a unique fragmentation pattern. Isobaric interference may be more substantial here because the pattern is not there If you add labeled amino acids to a cell culture you will get isotopic information in lots of places, and a library of labeled proteins. My guess is that analysis of such a mixture is probably challenging to sort out computationally because of all the different ways the labeled amino acids could be distributed within a given protein. But is very global, and yields a huge library of labeled proteins (consider the case of the 15 N labeled rat!), that is said to be less expensive per protein when using the proteins for subsequent isotope dilution experiments The authors seem to indicate that relative to ELISA and antibody or other biorecognition approaches MS will be slow they imply that the 2D chromatography is to blame and in my opinion the plethora of data that the MS yields must be a part of it. My counterpoint to this is that the MS experiment tells you everything (too much sometimes, as it may bury the important point) whereas the ELISA may only tell you about one thing (though, it is more likely to be what you asked) The authors are also skeptical of the quantitative accuracy of the protein ratios that isotope dilution analysis gives us. They say that as the ratio diverges from one (i.e. in case of a signal), the percent error increases They cite cost and time that make experiment replication difficult for assessment of error.

2 They note that a detailed analysis (they mean a statistical analysis of the labeling that will give a theoretical mass spectrum) of the type of label can help to estimate error levels, but that it is rarely done. I can see why it sounds complicated. Software is needed to help with this approach to getting a standard deviation estimate. 2. Supplementary Information: See Web

3 3. Questions 3.1. Define resolution for MS and for chromatography. Resolving power or resolution may be defined for one peak relative to another or simply based on a single peak What is an internal standard and what does it accomplish analytically Does an internal standard account for losses during sample preparation? If so, how? 3.4. Discuss isotope dilution from the perspective of a perfect internal standard Distinuish between a stable isotopic label and a radioisotopic label. Why are stable isotopic labels used in MS?

4 3.6. Define proteomics Define polynucleotide, polypeptide and protein Would you describe the contents of a cell as: A solution of one or two different proteins A solution of a few polynucleotides and polypeptides A solution with a few mono and polysaccharides, lipids, glycolipids, proteins, glycoproteins, peptides, RNA, DNA, small molecule electrolytes and metabolites and basically a very complex thing How does chromatography help to simplify the analysis of a cell s contents? How does mass spectrometry help?

5 Questions that are more specific to MS: How is product ion scanning superior to single quadrupole analysis of a protein mixture for the purpose of identifying or quantifying a given component of a complex biological sample? Given the exact structure of a small molecule, say, CO, and the mass spectrum comprising just the monocation, CO+, how many peaks do you expect to find in the MS of this molecule? Assume you can measure within about 100:1 signal to noise ratio. If more than one, then with what relative intensities? Apply the same reasoning to the analysis of a 5 kda protein (not in detail, just discuss the general idea).

6 3.14. Contrast quadrupole MS detection with fluorescence and absorbance spectroscopy in terms of: Figure of Merit q-mass Spec Fluorescence Absorbance Sensitivity Selectivity Generality Destructive or non? Sample Size Required Speed Cost Instrument size

7 Questions we have answered: Chem 250 Fall 2008 Answers to questions given in class A strong cation exchange resin contains sulfonic acid (a strong acid) residues. Assume the solution ph is Will all proteins bind to this resin? No, a cation exchange resin has anionic (strong acid) groups on the surface such as R- SO3 -. Therefore it will bind proteins that are either net cationic or that have at least some patches of positive charge on the surface. It will not bind purely anionic proteins. It will not bind proteins with a pi above the buffer ph = How will proteins bind to this resin? Electrostatic attraction between cationic (lysine and arginine) groups on the protein s periphery and sulfonates on the resin Will this be a mono- or multi-valent binding interaction? (Note that multivalent interactions such as DNA duplexation have characteristically sharp association isotherms.) The likelihood is that this will be multivalent. The more lysine groups on the protein surface, the more points of attraction and the stronger the binding Why would increasing ionic strength of, e.g. NaCl decrease the strength of this binding interaction? Increasing ionic strength will tend to elute the protein. One can think of this as a competitive binding effect (like the Na + in the solution competes with lysine + for the sulfonate) or as a charge screening effect wherein at high ionic strength ions in solution simply tend to cancel electric fields and prevent the normal electrostatic interactions from extending beyond a few angstroms On what basis will this separation work? The separation will work on the basis of the strength of the cation-anion attraction balanced against the solvating (enthalpic) and liberating (entropic) energies of releasing the protein On what basis does reverse-phase chromatography separate analytes? Hydrophobicity or, the tendency of non-polar components of the solution to stick to or partition into the oily coating on the chromatographic stationary phase.

8 3.17. Will ionic strength have a dominant influence on reverse-phase chromatographic separation carried out in 100% aqueous buffer? Ionic strength will only slightly modify the retention in the reverse phase system compared to the hydrophobic/hydrophilic quality of the protein (solute / analyte). Even if it does change the retention properties, the idea is that the basis of the separation on the reverse phase column will be different to that of the ion exchange column How does the combination of strong cation exchange followed by reversephase LC equate to a 2-D separation? Just sketch the outcome. absorbance t R / min μ = 0.01 μ = 0.05 Ionic strength (μ) / M μ = 0.1 μ = 0.5