*4.7. Normalization of Protein Abundance*

To correct for sampling variations resulting from random spot urine collection, the raw LFQ values for each protein were divided by the amounts of total protein and creatinine in each sample, followed by normalization of the corrected LFQ values by endogenous proteins without spike-in standards [59]. To identify endogenous urinary proteins for normalization, the 112 initial completely quantified proteins were considered, with six selected based on the following criteria: (1) quantified in all 54 samples; (2) corrected LFQ values did not differ significantly in the poor and good prognosis groups by the Mann–Whitney U Test (*p*-value > 0.05); and (3) had nearly persistent urine concentrations throughout the sample as top-ranked by NormFinder stability value [60].

The corrected LFQ values of the six selected normalization proteins in each sample were divided by their median value in all samples. The median of these six ratios was defined as the normalization scaling factor (NSF) for that sample. For example, NSF for sample s can be determined using the equation:

$$NSF\_5 = median \left(\frac{N\_{1,s}}{\hat{N}\_1}, \frac{N\_{2,s}}{\hat{N}\_2}, \dots, \frac{N\_{6,s}}{\hat{N}\_6}\right) \tag{1}$$

where *Ni*, *<sup>s</sup>* is the corrected LFQ value of normalization protein i in sample s and *N*ˆ*<sup>i</sup>* is the median corrected LFQ value of normalization protein i in all samples. Except for the six normalization proteins in a sample, the normalized LFQ value of each protein was calculated by dividing its corrected LFQ value by NSF.

$$LFQ\_{j,s} = \frac{LFQ\_{j,s}}{NSF\_s} \tag{2}$$

where *LFQ*˘ *<sup>j</sup>*,*<sup>s</sup>* is the normalized LFQ of urinary protein j in sample s and *LFQj*,*<sup>s</sup>* is the corrected LFQ of the corresponding protein [61].
