Label-free quantitation

However, this method is not recommended for low-abundance proteins due to poor accuracy. Software to assist in label-free quantitation includes an ion intensity chromatogram measurement called MSI filtering. This increases peak selection accuracy by incorporating the peptide retention time... 

Ouyang H, DeLouise LA, Miller BL, Fauchet PM (2007) Label-free quantitative detection of protein using macroporous silicon photonic bandgap biosensors. Anal Chem 79 1502-1506... 

Suter JD, White IM, Zhu H, Shi H, Caldwell CW, Fan X (2008) Label-free quantitative DNA detection using the liquid-core optical ring resonator. Biosens Bioelectron 23 1003-1009... 

Label-free quantitation is a rapid and fairly accurate approach for the survey of change in protein expression levels between two biological samples and is a promising alternative to stable isotope labeling approaches. It is a highly reproducible method that enables relative quantitation of the proteins across samples [14,15]. In a label-free quantitative approach, each sample has to be analyzed individually and sequentially by MS (unlike... 

Spectral Count The total number of MS/MS spectra (usually corresponding to redundant and nonre-dundant peptides) used for identification of proteins. Spectral count increases with protein abundance, hence spectral counting is a useful approach in assessing relative protein abundance by a label-free quantitative strategy. 

Zhu, W, Smith, J.W, Huang, CM. (2010) Mass spectrometry-based label-free quantitative proteomics. Journal of Biomedicine and Biotechnology, 2010, 840518. 

M.N., Hu, C.D., and Cheng, J.X. (2010) Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegans. /. Lipid Res., 51, 672-677. 

Neilson, K.A., Ah, N.A., Muralidharan, S., Mirzaei, M., Mariani, M., Assadourian, G., Lee, A., van Sluyter, S.C. and Haynes, P.A. (2011) Less label, more free approaches in label-free quantitative mass spectrometry. Proteomics 11, 535-553. 

Another label-free optical detection method—FTIR-ATR—has been applied for detection of thrombin by means of DNA aptamers [73], The antithrombin DNA aptamer previously developed by Tasset et al. [17] was immobilized covalently onto Si surface using UV irradiation method. As a quantitative measure, the area of N-H and CH2 bands was used. This method allowed to detect thrombin with a sensitivity around 10 nmol/L. The specificity of binding of protein to aptamer was also investigated using DNA with no binding site for thrombin. It has been noted that for effective binding study by FTIR-ATR method, the concentration of protein should be kept lower than 100 nmol/L. 

Various quantitative and statistical validation processes have been described, accounting for the fact that SpCs tend to be small numbers and vary due to the partial stochasticity of the process. In the example datasets included in this chapter, the relationship between the SpC and protein abundance obtained experimentally is shown in Figure 2, demonstrating that many proteins in bacterial cells are low in abundance while a small subset are highly abundant. Several studies have compared label-free with labeling methods or assessed its statistical validity (93-95). Overall, SpC alone should not be used as a means for absolute quantification (92,96), but it is quite adequate for... 

With the label-free methods in the repertoire of quantitative proteomics practitioners, there is still a need for label-assisted methods to perform quantitation with improved accuracy and precision. Label-assisted methods incorporate stable isotope labels into internal standards (IS) for quantitation this MS-based quantitation method is termed stable isotope dilution. The incorporation of stable isotope labels can be achieved either with or without... 

Co-immunoprecipitation is a common technique to enrich protein interaction complexes. This method uses an antibody to bind a known protein, which in turn has known or unknown binding partners. Shotgun proteomics is then performed to determine the enriched proteins. Quantitative MS is used to validate bound proteins and measure binding stoichiometry for proteins in the complexes prepared via IP (127,128). Quantitation approaches used include SILAM (129), label-free (130), and AQUA peptides with a normalization step (131). 

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. 

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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