Anchorchip

Three biological experiments were carried out with three technical replicates. The total number of samples analyzed by MALDI was 300. The number of technical replicates for protein identification by MALDI mass spectrometry was 2-3 (up to 5 for important and low-abundance proteins). Individual protein spots, selected on the basis of image-analysis output, were excised and digested in-gel with trypsin (Trypsin V511, Promega, Madison, WI, USA) as previously described [19 (link)]. For MALDI-TOF identification, 0.5–1 μL of the sample (50% solution of acetonitrile in water, 0.1% TFA) was placed on a ground steel MALDI target plate or AnchorChip or SmallAnchor (depending on the protein quantity), and 0.5–1 μL of the matrix (α-cyano-4-hydroxycinnamic acid) (Bruker Daltonics, Bremen, Germany) was added.

MALDI-TOF was employed to identify the proteins which showed reactivity. In-gel, digestions of spots were done according to the protocol reported by Shevchenko et al. (1996) (link). The procedure was adopted from our earlier study (Singh et al., 2015 (link), 2018 (link)). In brief, an automated robotic enzymatic (Trypsin) digestion of spots of interest was done by using a protein digester (Model Investigator ProPrep, Genomic Solutions Ltd., UK) and was further purified by using ZipTipC18 (Millipore). The purified proteins were then applied to AnchorChip (Bruker) with 2 μl of the matrix [a saturated solution of α-cyano-4-hydroxycinnamic acid (HCCA) made in 50% ACN and 0.2% TFA]. Mass spectra of digested peptides were acquired by Autoflex II TOF/TOF50 (Bruker Daltonik GmgH, Leipzig, Germany) in positive reflectron mode and the detection range of 500–3,000 m/z. The peptide mass fingerprints were searched by using the Mascot Wizard program (Matrix Science, Ltd., London, United Kingdom). Search parameters used in MS/MS for the identification of spots were peptide mass tolerance ±0.5 Da ppm, peptide charge state 1+, and maximum missed cleavages one.

Peptide profiles were analyzed with an ultraflex TOF/TOF (Bruker Daltonics, Massachusetts,USA). The samples were desalinated and concentrated using ZIP-TIPTM (Millipore, Perkin Elmer, UK) and then mixed with a-cyano-4-hydroxycinnamic acid matrix solution according to the proportion of 1:1. The mixture was deposited in the AnchorChip (Bruker Daltonics, Massachusetts,USA) for future use. The reflex pattern was automatically controlled by the FlexControl software. The Mascot software (Matrix Science Ltd, London, UK) was used to analyze the PMF and LIFT MS peak. The BioTools software (Bruker Daltonics, Massachusetts,USA) was used to search and identify PMF and MS/MS data with a peptide mass tolerance of 100 ppm. Protein identifications were accepted when the observed and predicted isoelectric points and relative molecular weights were consistent, and scores indicated nonrandom identifications at a significance level of P < 0.05.

Three biological experiments were carried out with three technical replicates. The total number of samples analyzed by MALDI was 150. The number of technical replicates for protein identification by MALDI mass spectrometry was 2–3 (up to 5 for important and low-abundance proteins). Individual protein spots, selected on the basis of the image-analysis output, were excised and digested in-gel with trypsin (Trypsin V511, Promega, Madison, WI, USA), as previously described [49 (link)]. For MALDI-TOF identification, 0.5–1 µL of the sample (50% solution of acetonitrile in water, 0.1% TFA) was placed on a ground steel MALDI target plate, AnchorChip, or SmallAnchor (depending on the protein quantity), and 0.5–1 µL of the matrix (α-cyano-4-hydroxycinnamic acid) (Bruker Daltonics, Bremen, Germany) was added.

The eluted sample was diluted 1:10 in matrix solution α-cyano-4-hydroxycinnamic acid (CHCA, 0.3 g/L in 2:1 ethanol:acetone), which was prepared daily. For analysis, 1 μL of the mixture was spotted onto an AnchorChip (Bruker Daltonics, Bremen, Germany) target and the droplet dried at room temperature before analysis.

The procedure of serum peptides enrichment and MALDI-TOF data acquisition were described previously [14 (link)]. The serum peptides were fractionated using weak cation exchange magnetic-beads (MB-WCX) (ClinProt purification reagent sets; Bruker Daltonics, Bremen, Germany) with a magnetic separator. Mixture with the eluted sample and matrix was spot onto a MALDI-TOF mass spectrometry target (AnchorChip™, Bruker Daltonics) for peptide profiling acquisition in Microflex mass spectrometer (Bruker Daltonics). Cilnplot standard was used for mass calibration. The scan range was 0.7–10 KD. Peptide profiling by different mass to charge ratio was obtained through FlexControl2.2 software. The coefficient of variability less than 30% indicated that the system was stable and reliable.