Supplementary Methods
SELDI profiling
CM10 chips were prepared and samples applied as previously described (Banks et al.
, 2005) but using 50 mM HEPES/0.1% v/v Triton X-100, pH 7.0 as the wash/dilution buffer. Chips were analysed using a PBS-II SELDI-TOF (Ciphergen) using a protocol with high mass acquisition of 200 kDa, optimised centre of 6.5 kDa, laser intensity of 175-195 and detector sensitivity of 9, collecting 50 transients per spot. External calibration was carried out using 400 fmoles of each of bovine insulin
(M.W. 5733.6), bovine ubiquitin (M.W. 8564.8), cytochrome C (M.W. 12230.9) and equine myoglobin (M.W. 16951.5) for data analysis which focussed on the m/z region between 1000-20,000 Da, with both the single and doubly charged peaks for each calibrant being used. All serum samples were analysed in duplicate with each replicate of each sample being randomised both in terms of the chip used and the day of experiment. For quality control purposes during the runs, an experimental QC sample (a serum pool prepared from 20 RCC and 20 normal serum samples) was also included on each chip, assigned to spot A for the first chip, spot B for the second and so on to ensure representative coverage of each spot. Peak detection was carried out using in-house software as previously described (Rogers et al.
, 2003). All samples and QC spectra were subjected to a range of QC analyses using in-house software (Cairns et al.
, 2009) and samples identified as outside tolerance levels were rerun.
Purification and Identification of Peak 1528 Da
Serum was thawed, vortexed, diluted 4x with 25 mM HEPES, pH 6.8 and injected onto a BioSuite CM 10µm, CXC cation exchange column previously equilibrated in
25 mM HEPES, pH 6.8 (Buffer A) using three consecutive 0.225 ml injections. The column was washed with 2.5 ml of buffer A and the bound material eluted with a 40

minute gradient to 100% Buffer A + 1.0M NaCl, at 0.5 ml/min, collecting 0.5 ml fractions. This chromatographic procedure was repeated and equivalent fractions pooled. At this stage and throughout the process, presence of the biomarker peak was monitored by desalting and concentration of a 15 µl aliquot of each fraction using C18 zip-tips and profiling by SELDI using gold chips and saturated sinapinic acid.
Fractions containing biomarker peaks of interest were concentrated and desalted using Bond-Elut C18-EWP cartridges. The cartridges were first washed with 2 ml of acetontrile and then equilibrated with 20 ml of 0.1% (v/v) TFA. Fractions were diluted with 0.1% (v/v) TFA to 10 ml final volume and recirculated over the Bond-Elut C18 cartridges 5 times using a vacuum manifold. The C18 cartridges were then washed with 10 ml of 0.1% (v/v) TFA and the bound proteins eluted with 1.0 ml of 90% acetonitrile/0.1% TFA prior to concentration in a speed vac to ~5 µl final volume.
Each fraction was then incubated with 50 µl of 50 mM ammonium bicarbonate, 10 mM DTT for 30 minutes at 45ºC, then a further aliquot of 50 µl 50mm ammonium bicarbonate, 55 mM iodoacetamide was added and a further incubation of 45 minutes performed at room temperature in the dark. Fractions were then concentrated by speed-vac to 10 µl volume, desalted using C18-zip-tips and eluted using 2.0µl of a freshly-prepared 5 mg/ml solution of 4-hydroxy
-cyano-cinnamic acid in 50% aqueous (v/v) acetonitrile/0.1% (v/v) TFA and applied to a 384-well
MALDI plate.
Positive-ion MALDI mass spectra were acquired over a mass range of m/z 800-4000 obtained using a Bruker Ultraflex III in reflectron mode with external calibration using des-Arg 1 -Bradykinin, 904.681; Angiotensin I, 1296.685; Glu 1 -Fibrinopeptide B,
1750.677; ACTH (1-17 clip), 2093.086; ACTH (18-39 clip), 2465.198; ACTH (7-38 clip), 3657.929). Peaks of interest were manually selected for MS/MS fragmentation.

The default calibration was used for MS/MS spectra, which were baseline-subtracted and smoothed (Savitsky-Golay, width 0.15 m/z, cycles 4); monoisotopic peak detection used a SNAP averaging algorithm (C 4.9384, N 1.3577, O 1.4773, S
0.0417, H 7.7583) with a minimum S/N of 3. Bruker FlexAnalysis software was used to perform the spectral processing and peak list generation. Tandem mass spectral data were submitted to database searching against the NCBInr protein database (2 nd
July 2008) containing 194674 human sequences using Mascot (Matrix Science Ltd., version 2.1) with search criteria including: Enzyme - none; Taxonomy - Homo sapiens; Variable modifications - oxidation (M); Peptide tolerance - 200 ppm; MS/MS tolerance - 0.8 Da; Instrument - MALDI-TOF-TOF. The search criteria also included
Carbamidomethyl (C) as a fixed modification for all alkylated samples.
Immunoprecipitation/SELDI-TOF-MS Studies
50 µl of protein G-Dynabeads were used per immunoprecipitation. The beads were washed four times with 0.5 ml of 0.47% (w/v) citric acid, 0.92% (w/v) sodium phosphate, pH 5.0 (antibody-binding-buffer) using a magnetic separator and then resuspended in a total volume of 0.5 ml of antibody-binding-buffer containing either 2
µg of mouse anti-human SAA Ab or 2 µg of mouse MOPC-21 antibody (negative control) and mixed end-overend overnight at 4°C. The next day the beads were washed three times with 0.5ml of antibody-binding-buffer eac h time and then 80µl of phosphate-buffer saline (0.2M Sodium-Phosphate, pH 7.4, 1,5M NaCl)) and 20 µl of donor serum added to each immunoprecipitation. The immunoprecipitations were then incubated end-overend overnight at 4°C and the next day the beads were washed three times with 0.5 ml of PBS each time. Antigen elution was performed by incubating the washed beads with 20 µl of 100 mM acetic acid for 10 minutes at room temperature and the supernatant removed after the beads were precipitated by magnetic separation. The acetic acid elate was then applied to 0.6 µl C18-zip-tips which had been pre-treated by one wash in methanol followed by extensive

equilibration with 0.1% (v/v) TFA. The zip-tips were washed five times with 20 µl of
0.1% (v/v) TFA each time and the bound species eluted onto Bio-Rad SELDI-gold chips using 2 µl of a saturated solution of sinappinic acid (SPA) in 60% (v/v) acetonitrile/0.04% TFA (for TOF-MS in the mass range 5-20kDa) or 2 µl of saturated
α-cyano-4-hydroxycinnamic acid (CHCA) in 60% (v/v) acetonitrile,/0.04% TFA (for
TOF-MS in the mass range 1-5kDa). The chips were then allowed to air-dry for 15 minutes and spectra collected using a PCS 4000 Enterprise, Protein Chip Reader
(Bio-Rad, Herts, UK) according to an automated data collection protocol for a molecular weight range of 0-40,000 Da. The following settings were used: (a) laser intensity of 1,500 nJ; (b) focus mass 65000 Da; (c) matrix attenuation 1000 Da; (d) sampling rate 400 MHz; (e) 2 warming shots (not included in analysis), 10 data shots per point and (f) total number of points evaluated equal to 12.5% of the spot surface.
Mass accuracy was calibrated externally using the all-in-one peptide standard according to the manufacturer’s protocol (Bio-Rad, Herts, UK). Peaks were detected after baseline subtraction and spectral calibration.