The absence of an internal standard spiked early in sample preparation protocols means that label-free methods are sensitive to technical variance, consequently, label-free proteomics requires high instrument performance and standardization of sample preparation methods.
Internal standardization (standard addition with spiked samples) was employed.
For internal standardization, a parallel real-time PCR targeted an invariant region in the converse exon within the same first-round amplicon template.
Also additives (in liquid or solid form) can be introduced for internal standardization, quantification (e.g., for standard addition or isotope dilution purposes), or to affect laser sample interaction (i.e., increased energy absorption at the applied laser wavelength).
As no internal standardization is possible in an experiment using density gradient fractions, these data were then normalized to the average of all evaluable data points.
In targeted analysis, SIL reference standards can be used for internal standardization of peak intensities in trace analysis by use of LC HRMS or LC–(HR MS MS [ 27, 28], making this the most common use of SIL.
Furthermore, performing real-time PCR-SSP in a multiplex format allowed the incorporation of internal standardization, measuring in parallel the degree of sequence identity between template and primers, and the amount of template incorporated in the reaction.
29Si was used for internal standardization and data were calibrated against two standard reference materials (NIST SRM 610, 612) and Corning B, C and D. The elemental compositions were then calculated according to Gratuze [38].
Concerning short-term repeatability external calibration with internal standardization using Rh was superior with an RSD of 0.9% which demonstrates the potential of FI-ICP-SFMS compared with FI-FLD, where an RSD of 6.5% was observed.
