Samples were meanwhile dried down to a final volume of 15 uL in vacufuge and desalted using ZipTip uC 18.Eluted samples were stored at ?20 C until use.Mass analysis Mass analysis was performed at Genome Research Centre at the University of Hong Kong using a 4800 MALDI TOF TOF analyzer.In house MASCOT search engine was used to analyze Mass Data.Data were BLASTed against both NCBInr and SwissProt modification,carbamidomethyl,vari able modification,oxidation,MS MS fragment tol erance,0.2 Da,precursor tolerance,75 ppm,peptide charge,1,monoisotopic.MASCOT cut off scores were set to 30.Only the peptides ranked first with p values smaller than 0.05 were accepted.Results Affinity chromatography was performed to find cellular targets of crocin in different organs.

There are two types of interactions between stationary phase and cellular proteins in affinity chromatography,specific interaction between crocin and its targets or unspecific binding of proteins to other parts of stationary phase such as agar ose beads.To reduce unspecific binding of non Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries target proteins,tissue extracts were incubated with control agarose beads.Unbound proteins were incubated with crocin resin beads.Target proteins were eluted using 2 M NaCl in binding buffer and subjected to 2D gel elec trophoresis.After in gel digestion of protein spots,MALDI TOF TOF was employed for their identification.Mass data were analyzed using MASCOT.Crocin resin conjugation Crocin was covalently attached to diaminodipropyla mine side chain of agarose beads using Mannich reac tion.Briefly,formaldehyde reacts with primary amino group to produce highly reactive iminium group.

This group can react with active hydrogen on hydroxyl groups of sugar residues on crocin.Yield of crocin con jugation to agarose beads was calculated to be 70%.In FT IR spectrum of crocin resin conjugate,hydroxyl groups of crocin glycosides were observed at 3410.06 cm 1 which is identical with that of pure crocin.Besides other peaks in the crocin resin con jugate Inhibitors,Modulators,Libraries were overlapped with those of pure crocin,sug gesting the presence of crocin with its functional groups within the conjugate.Target proteins of crocin in kidney Beta actin like protein 2,cytochrome c1,proteasome subunit alpha type 6 and proteasome subunit alpha type 4 were identified as cellular targets of crocin Inhibitors,Modulators,Libraries in kidney.

Target proteins of crocin in heart Data indicated that crocin binds to mitochondrial ATP synthase Inhibitors,Modulators,Libraries subunit beta,beta actin like protein 2,cyto compound library chrome b c1 complex subunit 1 and subunit 2,and acetyl CoA acetyltransferase in heart.Target proteins of crocin in Brain Target proteins of crocin in brain were identified as tubulin beta 3 chain,tubulin beta 6 chain,mitochondrial ATP synthase,beta actin like protein 2,14 3 3 protein beta alpha,tyrosine 3 monooxygenase,V type proton ATPase catalytic subunit A,60 kDa heat shock protein,creatine kinase B type and peroxiredoxin 2.