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Background: The gene mutations found in children with cystinuria cannot solely explain kidney stone formation, suggesting that specific proteins may serve as promoters of cystine precipitation, aggregation or epithelial adherence. We aimed to identify proteins unique to cystinuria and kidney stone by performing urine proteomic analysis.
Methods: We compared urinary proteomes of 2 children with cystinuria and kidney stones and 2 healthy children (measurements performed in duplicate), using mass spectrometry (MS) total ion current quantification of proteins. Proteins of interest were selected based on the number of assigned peptides that map to a particular protein (more than 5), and unique peptides that have been identified for each protein (more than 2) as a threshold to be well above observed technical variations in MS experiments.
Results: Of the 665 proteins identified by proteomic analysis, 152 proteins were found only in children with cystinuria and kidney stones, 12 of which met the selection criteria (Table). Additionally, these children were found to have 2-4 times higher urinary levels of ceruloplasmin, and lower levels of osteopontin and uromodulin compared to controls.

Accession Number*	Protein	MW (kDa)	No. of assigned peptides**	No. of unique peptides**	Sequence coverage (%)***
ANXA1	Annexin A1	39	14/12	9/8	32/29
FIBB	Fibrinogen beta chain	56	129/91	25/21	66/49
ITIH2	Inter-alpha-trypsin inhibitor	106	33/81	12/11	27/16
TRFL	Lactotransferrin	78	185/11	36/3	60/6
MMP9	Matrix metalloproteinase 9	78	26/15	9/7	18/12
PERM	Myeloperoxidase	84	88/6	19/5	32/3
PLSL	Plastin-2	70	42/8	14/4	33/11
PON1	Serum paraoxonase	40	23/74	9/14	48/55
TBA1B	Tubulin alpha-1B chain	50	9/13	8/4	27/15
VIME	Vimentin	54	10/9	8/4	21/14
NGAL	Neutrophil gelatinase-associated lipocalin	23	110/40	10/6	66/45
TAGL2	Transgelin-2	22	13/7	6/4	50/33

*Human; **Values presented in each patient
*** % of sequence that has been identified for each protein
Conclusions: Children with cystinuria and kidney stones have a different urinary polypeptide profile compared to healthy controls. These unique proteins merit further investigation. Proteomic analysis may be useful to identify potential disease biomarkers and novel therapeutic targets.