Label-assisted 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... 

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... 

Chen, Q Wu, J., Zhang, Y, Lin, J.-M. (2012) Qualitative and Quantitative Analysis of Tumor Cell Metabolism via Stable Isotope Labeling Assisted Microfluidic Chip Electrospray Ionization Mass Spectrometry. Anal. Chem. 84 1695-1701. 

O. A. Mirgorodskaya, Y. P. Kozmin, M. I. Titov, R. Kbmer, C. P. Sonksen, and P. Roepstorff. Quantitation of Peptides and Proteins by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Using lsO-Labeled Internal Standards. Rapid Commun. Mass Spectrom., 14(2000) 1226-1232. 

In this chapter, we provide protocols to determine the ability of a peptide to mediate DNA internalization in cultured human tumor cells. Fluorescence-assisted cell sorting (FACS) analysis is used to obtain quantitative data on the time and temperature dependence of macromolecular delivery. Confocal microscopy is used to study the subcellular localization in both fixed and live cells. Fluorescently labeled transferrin and dextran are used to label the clathrin-dependent (15) and the non-clathrin, non-caveolar (16) endocytic compartments, respectively. Expression of a caveolin-l-YFP fusion protein is used to label cell surface caveolae and intracellular caveosomes (17). Finally a protocol, for the overexpression of dominant-negative dynamin [GTPase deficient dynamin-2 containing the amino acid substitution K44A (18)] is provided to evaluate the dynamin dependence of the uptake mechanism. 

Figure 8 Scheme for differential labeling of two samples with do/da acrylamide (alkylation of Cys residues). The central map refers to rat sera, labeled separately with either do- or ds-acrylamide and mixed in a 30 70% ratio. (Reproduced from Gehanne S, Cecconi D, Carbon L, et al. (2002) Quantitative analysis of two-dimensional gel-separated proteins using isotopically marked alkylating agents and matrix-assisted laser desorption/ionization mass spectrometry. Reipid Communications in Mass Spectrometry 16 1692-1698.)... 

Label-free methods use the intrinsic properties of proteins to report binding events on protein chips. Ciphergen ProteinChips can be incorporated directly into a matrix-assisted laser desorbtion/ionization (MALDI) source of a mass spectrometer, which can identify the proteins and provide quantitative analysis. The ionization of proteins bound to Protein-Chips is enhanced by the properties of the chip surface, leading to more uniform mass spectra than possible with standard MALDI mass spectrometric... 

Titov, M.I., Komer, R., Sonksen, C.P., and Roepstorff, P. (2000) Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)0-labeled internal standards. Rapid Commun. Mass Spectrom., 14,1226 1232. 

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