4800 maldi tof tof

MALDI-TOF experiments and analysis were conducted at the Rutgers Biological Mass Spectrometry Facility. Samples for both shrimp and pig tropomyosin were prepared as described above, and subject to simulated gastric digestion in pepsin for 0, 10, 30, 60, 120 and 240 minutes. Subsequent intestinal digestion was performed for 30 minutes starting with gastric 60 or 120 digestion end-products. Samples were brought to a pH of 8–9 and final concentration of 0.33 mg/mL for 0, 10, 30 and 240 min gastric digestions, or 0.30 mg/mL for gastric/intestinal digestions of 60 or 120 minutes. Prior to analysis, samples were 1:5 diluted with matrix (10mg/ml sinapinic acid in 50% acetonitrile, 0.1% trifloroacetic acid) and deposited on an opti-TOF 384 well insert for MALDI-TOF/TOF (ABSciex) using dry-droplet method. The MALDI-TOF data were acquired using 4800 MALDI-TOF/TOF (ABSciex) with linear mid mass positive mode. Data were acquired from 3kDa to 40 kDa mass range with external calibration by apo-myoglobin singly charged, doubly charged ions as well as dimer ions. The laser was fixed at 5.6 kV, and spectra were based on accumulation of 1000 laser shots. Peaks with a minimum signal to noise ratio of 10 were exported from the Applied Biosystems 4000 Series database into mgf files using the TS2 Mascot utility.

To analyse the mass of B. subtilis proteins co-purifying with ClpP(6His) under heat-shock conditions (Extended Data Fig. 1), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF-MS) was performed. The corresponding protein purifications were spotted on a MALDI plate using a sinapinic acid (10 mg ml⁻¹) matrix prepared in 50% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA). The samples were measured in a 4800 MALDI-TOF-TOF (AB Sciex) instrument operated in linear mode. Calibration was performed internally using cytochrome C as standard.

Among the spots with a change in %volume with IVIg therapy, some protein identifications were made with a 2DE CSF map [19 (link)], others were made by using tryptic digestion followed by MS. The details of the digestion and MS protocols have been previously published [20 (link)]. The MS analysis was done using a 4800 MALDI TOF/TOF (AB Sciex, Framingham, MA). Peptide mass fingerprint data were collected in positive ion reflector mode in the range of 900 to 4000 mass to charge ratio. Several of the highest intensity non-trypsin peaks were then selected for tandem mass spectrometry analysis. MS/MS was collected in positive ion mode with default calibration. PMF and MS/MS spectra were analyzed using GPS Explorer (version 2.0, AB Sciex), which acts as an interface between the Oracle database containing raw spectra and a local copy of the Mascot search engine (version 2.0, Matrix Science, London, United Kingdom). Data were searched against a locally stored copy of the NCBInr protein database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein&itool=toolbar). A mass tolerance of 25 ppm was used for the PMF data and 0.2 Da for the MS/MS data. For a database match to be considered a positive identification, a value of p < 0.05 (calculated by GPS Explorer) was required in addition to at least one high confidence (95% confidence interval or higher) MSMS match.

The morphologies of Fe₃O₄ NPs, CNPs, and CNPs@Fe₃O₄ NCs were characterized by using transmission electron microscopy (TEM, Hitachi H-8100). UV-Vis spectra were recorded using Shimadzu UV-1800 spectrophotometer. Powder XRD analyses were performed on a Rigaku Ultima III diffractometer operating with a Cu-Ka radiation source filtered with a graphite monochromator (λ = 1.5406 Å). A 4800 MALDI TOF/TOF (AB SCIEX) equipped with a pulsed Nd:YAG laser at an excitation wavelength of 355 nm, was used for the analyses. For each MS spectrum, 1250 laser shots were fired with a total of 50 sub-spectra and 25 shots per subspectrum. Data Explorer 4.9 software (AB SCIEX) and GlycoWorkbench software were used for MS data interpretation and glycan analyses.