Relative quantification methods peptide

Relative quantification methods provide information on the identity of peptides/proteins in a sample as well as their levels expressed in amounts relative to each other. Some of these methods rely on label-free strategies while others incorporate stable isotopes into one or more of the samples, allowing them to be combined and analyzed together. In both cases, the strength of the signal for each peptide is a reflection of the amount of peptide present in the sample, providing quantitative information. 

Fig. 25.8. Differential images of the distribution of peptides with simiiar moiecuiar masses (peptide ion images extracted at m/z 0.0025 Da). Note that these images demonstrate the distributions of singie isotopes i.e., the 1,011.410 moiecuiar species is clearly resolved from the second isotopes of 1,010.472 and 1,010.590 (respectively 1,011.472 and 1,011.590) (see Note 7). The higher relative intensity of the 1,011.410 signal vs. the 1,010.472 signal is in good agreement with previously detected by different methods variety In abundances of these peptides (Pea-CAH-I is more than twice as abundant as Pea-CAH-ll, 12) (see also Note 9 on relative quantification).

Total protein assays have the advantage of being relatively straightforward compared to molecular-level analyses. Methods with fluorescence-based detection are also highly sensitive, and thus amenable direcdy to DON. Quantitative interpretation for environmental mixtures such as seawater, however, may be problematic for some samples. Most methods react with specific moieties (e.g., coomassie blue binds to lysine and arginine) and thus results obtained can depend on protein composition, size distribution, and even conformation (Sapan et ai, 1999), making the careful choice of calibration standards important. In addition, common components of natural samples, such as humic materials (e.g., Mayer et ai, 1986), carbohydrates (Sapan et ai, 1999), or NH3 may interfere with quantification. Overall, colorimetric methods can be very useful as quick, Hkely semi-quantitative estimates of total protein or peptide. However, potential biases inherent in the mechanism of a specific method should be considered before one is chosen, and appHcation of newer molecular assays (e.g., CBQCA) should be carefully examined in terms of natural sample matrix (Nunn et ai, 2003). 

As the name suggests, label-free quantification does not require the incorporation of an isotopic label into peptides for quantitation of relative concentrations. Each sample is separately prepared, subjected to individual LC/MS runs, and quantification is performed through analysis of their mass spectra. There are two approaches used for label-free quantification. The first approach is based on the assumption that peptide/protein levels follow a linear relationship with area under the curve of the ion spectra. This method involves quantitation of relative levels by comparing the peak intensity of ions from multiple peptides. But experimental variations in sample preparation and... 

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