*2.2.5. Selected reaction monitoring (SRM)/multiple reaction monitoring (MRM)*

One of the ways frequently used in replacement of shotgun MS/MS is selected reaction monitoring (SRM) [60], also known as multiple reaction monitoring (MRM). This assay makes use of a triple-quadrupole mass spectrometer [60], where peptides from a previously digested protein go through. In the first analyzer, molecular ions with similar mass to the peptide are selected, followed by fragmentation of the peptide bonds in the second analyzer. Lastly, in the third and final analyzer, the fragmented ions from the peptide pieces are measured, originating a transition signal [61]. This method does not record any full-mass spectrum, increasing its sensitivity and allowing the detection of scarce proteins in complex mixtures [60].

#### *2.2.6. Fourier transform infrared and Raman spectroscopy*

FTIR is a procedure that gives information about the surface properties of the nanoparticle and the protein corona, as it allows the detection of its attachment [33]. Standard Raman spectroscopy is not normally used independently, and it's usually meant to complement other methods, such as FTIR [62]. Together, they provide information about protein's secondary structure [63], plus vibrational and rotational parameters.
