30 kda filter

On a 30-kDa filter (Millipore, Billerica, MA), 100 μg of each of the patient samples and the super-SILAC mix were mixed. The samples were further processed by the FASP method in which the SDS buffer is exchanged with a urea buffer (21 (link)). This was followed by alkylation with iodoacetamide and overnight digestion by trypsin at 37 °C in 50 mm ammonium bicarbonate. The tryptic peptides were collected by centrifugation and elution with water (two times).
Strong anion exchange chromatography was used to fractionate 40 μg of peptides from each patient sample (22 (link)). It was performed in tip-based columns from 200-μl micropipette tips stacked with six layers of a 3M Empore anion exchange disk (1214-5012; Varian, Palo Alto, CA). For the fractionation, a Britton and Robinson universal buffer (20 mm acetic acid, 20 mm phosphoric acid, and 20 mm boric acid) was used and titrated using NaOH to six buffers with the desired pH values (pH 11, 8, 6, 5, 4, and 3). Subsequently, six fractions from each sample were collected followed by desalting the eluted fractions on reversed phase C₁₈ Empore disc StageTips (23 (link)). The peptides were eluted from the StageTips using 20 μl of buffer B composed of 80% ACN in 0.5% acetic acid (two times). A SpeedVac concentrator was used to prepare the samples for MS analysis by removing the organic solvents.

Approximately 10 mg of kidney tissue (n = 6 per group, Table 1) was lysed in RIPA buffer (Pierce) using a bead beater homogenizer (Precellys). A BCA assay was performed and 50 µg of total protein from each sample was taken for Filter Aided Sample Preparation (FASP) digestion. In short, 30 kDa filters (Millipore) were equilibrated with 8 M urea buffer and spun through. For protein reduction, 50 µg of tissue lysate was added to 100 µL of 20 mM dithiothreitol (DTT) and incubated for 30 min at room temperature (RT). Samples were alkylated in 100 µL of 100 mM iodoacetamide (IAA) and incubated for 30 min at RT and in the dark. The samples were centrifuged and buffer exchanged with 8 M urea twice, and then with 50 mM ammonium bicarbonate three times. Trypsin was added at an enzyme:protein ratio of 1:30 (50 µL of 0.3 µg trypsin), and incubated overnight at 37 °C. The following day, the filters were inverted, centrifuged, and the digested peptides collected with washing steps of 0.5 M NaCl, ensuring maximum yield. The digested peptides were desalted using SOLAµ™ cartridges, as per the manufacturer’s instructions. Eluted samples were dried using a vacuum concentrator (Speedvac, Eppendorf) and resuspended in buffer A, consisting of 98% MilliQ-H₂O, 2% acetonitrile (ACN) and 0.1% formic acid (FA). Samples were stored at − 20 °C until analysis by mass spectrometry (MS).

NPX encapsulation efficiency (% EE) of the formulations was determined by the ultrafiltration-centrifugation method, using 30 kDa filters (Millipore) and 4100 g, for 20 min. The non-encapsulated naproxen fraction was quantified by HPLC in a Varian ProStar (Agilent Technologies, USA) chromatograph, using a C18 column (Alcron Luna^®) at 25 °C. The mobile phase (1.8 mL min⁻¹ flow rate) was composed of acetonitrile:water:acetic acid (50:49:1 v:v). The amount of encapsulated naproxen was determined by subtracting the non-encapsulated (free) fraction from the total amount of naproxen (total NPX) in the sample prior to phase-separation, according to Eq. 1: $$\%EE=\frac{totalNPX-freeNPX}{totalNPX}\times 100$$
The samples (n = 3) were quantified immediately after preparation and after 12 months to evaluate the nanoparticle stability.

Tumour samples were subjected to lysis by sonication in 2% SDS buffer containing, phosphatase inhibitors such as 1 mM sodium fluoride, 2.5 mM sodium pyrophosphate and 1 mM sodium orthovanadate and 1 mM β-glycerophosphate. Subsequently, tissue lysates were obtained by centrifuging at 18,000 g at 4 °C for 20 min. Bicinchoninic acid (BCA) assay (Pierce, Illinois, USA, Cat #23,225) was performed to measure the protein amounts. Approximately 3 mg equivalent protein from each tissue was pooled in order to constitute a final protein amount of 15 mg in each NF-PitNET subgroup. The pooled lysates of four subgroups were subjected to reduction with 5 mM dithiothreitol (DTT) for 40 min at 60 °C and alkylation with 20 mM iodoacetamide (IAA) in dark for 15 min. Prior to proteolytic digestion, buffer exchange with 8 M Urea and 50 mM triethylammonium bicarbonate (TEABC) was carried out using 30 KDa filters (Millipore) and protein estimation was performed. Protein amounts were confirmed by normalization on SDS-PAGE across the four subgroups of NF-PitNET. Further, proteins were subjected to digestion with trypsin (Worthington Biochemical Corporation) for 16 h at 37 °C in 1:10 (w/w) ratio of enzyme to substrate. The efficiency of trypsin digestion was confirmed on a 10% resolving gel and continued with TMT labelling.