Labeling and Quantitation Problem Set

Labeling and Quantitation problem set

Lecture 13 CH908 Mass Spectrometry

1. It has been demonstrated that the chromatographic separation of deuterated and nondeuterated peptides by reversed-phase chromatography could cause substantial systematic errors in quantitative proteomics.

(A) Explain the mechanism of isotopic separation by of the labelled peptides by RP HPLC.

(B) Consider the three H/D labelling reagents:

- d0 and d3 forms of N-acetoxysuccinimide;

- d0 and d4 forms of succinic anhydride;

- d0 and d8 forms of the commercial ICAT reagent

Please estimate which label will exhibit the highest and the lowest degree of isotopic separation, and explain your estimations.

2. In the bottom-up quantitation approaches used in proteomics, chemical labelling can be performed either before or after enzymatic digestion. Either way has its advantages and disadvantages. Please state these. Consider the labelling efficiency, ease of sample cleanup, and the accuracy of quantitation.

3. The iTRAQ label, though considered “isobaric”, is actually a set of four compounds which have different isotopic composition, as follows:

Isotope / Mass
1H / 1.0078
12C / 12.0000
13C / 13.0034
14N / 14.0031
15N / 15.0001
16O / 15.9949
18O / 17.9992

Calculate the exact mass difference between these compounds (consider the tag part and disregard the leaving group). Estimate resolution which would be necessary for baseline resolution of all 4 peaks in MS for 1000 Da and 2500 Da peptides. Is this resolution achievable using modern MS detectors? What type(s) of detector(s) would (possibly) allow such resolution?

4. (A) Calculate the mass defect for the mass defect labeled peptide from the J. Amster’s paper (Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses. H. Hernandez, S.Niehauser, S.A. Boltz, V. Gawandi, R. S. Phillips, and I. J. Amster, Anal Chem. 2006 May 15; 78(10): 3417–3423).

The MDL is 2,4-dibromo-(2′-iodo)acetanilide, and the labeling reaction is as follows:

Isotope / Mass
32 S / 31.9721
79 Br / 78.9183
81 Br / 80.9163
31 P / 30.9738

(B) Estimate the place of the MDL-modified peptide, M = 1500 Da, on the peptide MM distribution histogram below. Support your estimates by calculations. Online tools may be used for this; if you used the online tools, please indicate what was used.