Quantification Label-free

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... 

R., Nielsen, P.H., and Dueholm, M.S. (2015) Label-free quantification reveals major proteomic changes in Pseudotnonas putida FI during the exponential growth phase. Proteomics, 15 (18), 3244-3252. 

Label-free quantification can also be performed using MALDI-MS and MS/MS. 

Label-free quantification is a very attractive method, because there are no extra costs involved. Instead, there are much higher demands on the reproducibility of technical parameters, such as sample loading, chromatography, and mass accuracy, and more experimental replicates are needed. In the following sections, different approaches to label-free quantification will be discussed the extracted ion chromatogram (XIC), spectral counting (SC), the protein abundance index (PAI), and the LCMS method. 

Figure 10.1. Workflow generally applied In proteomic studies. Label-free quantification methods do not Include this step.

Electrochemical immunosensors are a powerful tool for the analysis of antibacterials in food and different configurations have been presented during recent years. For example, an amperometric immunosensor was reported by Wu et al. [182], for penicillin quantification in milk, with a linear range from 0.25 to 3 ng/ml and a limit of detection of 0.3 pg/L [182]. Other types of transduction have been also explored, like a label-free impedimetric flow injection immunosensor for the detection of penicillin G. 

LABELING AND LABEL-FREE APPROACHES FOR PROTEIN QUANTIFICATION... 

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... 

Schneider BH, Edwards J, Hartman N (1997) Hartman interferometer versatile integrated optic sensor for label-free, real-time quantification of nucleic acids, proteins, and pathogens. Clin Chem 43 1757-1763... 

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. 

Hiramatsu R, Matsumoto M, Miwa Y, Suzuki Y, Saito M, Miyazaki Y (2002) Characterization of Shiga toxin-producing Escherichia coli 026 strains and establishment of selective isolation media for these strains. J Clin Microbiol 40 922-925 Hu Y, Zhang Q, Meitzler JC (1999) Rapid and sensitive detection of Escherichia coli 0157 H7 in bovine faeces by a multiplex PCR. J Appl Microbiol 87 867-876 Huang L, Cooper MA (2006) Real-time label-free acoustic technology for rapid detection of Escherichia coli 0157 H7. Chn Chem 52 2148-2151 Ibekwe AM, Watt PM, Grieve CM, Sharma VK, Lyons SR (2002) Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli 0157 H7 in dairy wastewater wetlands. Appl Environ Microbiol 68 4853 862... 

Sohd supported membranes immobihzed on quartz resonators allow for the detection and quantification of protein-membrane and membrane-membrane interactions mediated by proteins. The advantage of the QCM technique is not only that a label-free monitoring of proteins is possible, but also that the good time resolution allows for a detailed quantitative analysis of kinetics, as demonstrated in this chapter by means of Monte Carlo simulations. Moreover, since the quartz oscillation is not only sensitive to... 

Panke, O., Kirbs, A., Lisdat, F. (2007). Voltammetric detection of single base-pair mismatches and quantification of label-free target ssDNA using a competitive binding assay. Biosens Bioelectron 22, 2656-2662. 

---