Ultraflex 3 mass spectrometer

Protein spots were automatically detected in the high-resolution digitized gel images and analyzed by Image Master 2D Platinum v 5.0 software (GE Healthcare). Well defined spots were manually selected, excised and processed as previously described [116 (link)]. Digestion was achieved with trypsin (Gold Mass Spectrometry Grade, Promega, Madison, WI) at 37°C, overnight.
Peptides from digested proteins were mixed with saturated solution of α-cyano- 4-hydroxy-cinnamic acid (HCCA) in 50% acetonitrile, 0.1% trifluoroacetic acid (TFA). The mixture was spotted onto a MALDI (Matrix Assisted Laser Desorption Ionization) target plate and allowed to crystallize at room temperature. The same procedure was used for the standard peptide calibration mixture I (Bruker Daltonics). For mass spectra acquisition, a MALDI-TOF-TOF (MALDI-time-of-flight in tandem) UltraFlex III mass spectrometer (Bruker Daltonics) was operated in the reflector for MALDI-TOF MS peptide mass fingerprint (PMF) and in the “LIFT™” mode for MALDI-TOF-TOF MS/MS fragmentation experiments, on fully manual mode using FlexControl software v. 2.2. To process the data obtained, Flex Analysis v.3.0 software (Bruker Daltonics, Billerica, MA) was employed.

2 μL of KRAS4b-FME (1 mg/ml) diluted with 8 μL of buffer containing 20 mM HEPES pH7.3, 100 mM NaCl, 5 mM MgCl₂, 0.5 mM DTT and added 1 μL of endoproteinase Glu-C dissolved in water at 0.5 mg/mL (Roche, Cat No 11 420 399 01). After incubation for 19 hours at room temperature (24 °C) in the dark 1 μL of the digest was mixed with 10 μL of DHB (2,5-dihydroxybenzoic acid) matrix (saturated in solution containing 10% acetonitrile, 0.1% trifluoroacetic acid), 2 μL of the mixture spotted on target plate and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) on Ultraflex III mass spectrometer (Bruker Daltonics) in reflector mode with 3000 laser shots acquired per spectrum (Fig. 1d). The m/z 2199.350 which was close to the expected m/z of C-terminal peptide of KRAS4b (KMSKDGKKKKKKSKKTKC) with carboxyl methylated S-farnesyl cysteine residue (expected m/z 2199.381) was analyzed by MALDI-TOF MS/MS. The spectrum information was processed using BioTools resident software (version 3.34) and compared with expected sequence KMSKDGKKKKKKSKKTKC with farnesyl and methyl groups on terminal Cys (C16H27, 218 mass units). Both N-terminal b - and C-terminal y–ions were identified confirming peptide identity (Supplementary Figure 4).

A total of 1 μl of purified GlyCer solution or peptide calibration standard (8206195; Bruker Daltonics) was sandwiched in 0.5 μl α-cyano-II-hydroxycinnamic acid on a matrix-assisted laser desorption ionization (MALDI) ground steel target (Bruker). Acquisition of MALDI spectra was performed using an ultraflex III mass spectrometer (Bruker Daltonics) in reflector mode, detecting positively charged ions in the range of 400 to 4,000 m/z. Each sample was analyzed for the expected signals at 778 m/z and 776 m/z for saturated GlyCer and unsaturated GlyCer, respectively. In addition, the instrument was operated in MS/MS mode for the parental ion masses of 777 and 778 as mentioned above. The evaluation of the spectra was carried out using the program “mMass—Open Source Mass Spectrometry Tool” (57 (link)).

Analyses were carried out with an Ultraflex^™ III mass spectrometer (Bruker), in positive reflector mode using a Nd:YAG laser operating at 355 nm. Spectra were acquired using flexControl 3.0 (Bruker) on a mass range of 700–3,500 Da with an accumulation of 500 shots on the standards and 1000 shots on the samples. The calibration standard (Bruker) was prepared according to the manufacturer’s instructions for instrument calibration and consisted of angiotensin I, ACTH clip(1–17), ACTH clip (18–39) and ACTH clip(7–38) peptides.

The points were transferred to new zip-seal storage bags and 2 mL of 0.6 M HCl was added to each of the original storage bags, as well as to one new bag which served as a blank control. The bags were heated at 65 °C for 4 hours, and the acid was pipetted out of the bags and neutralized using 0.1 M NaOH. The neutralized solution was freeze-dried to reduce the volume, and then re-suspended in 50 μL of 50 mM ammonium bicarbonate buffer (NH₄HCO₃, pH 8.0, AmBic); 0.4 μg of trypsin was added and the samples were heated at 37 °C for approximately 18 hours. The samples were acidified to 0.1% trifluoroacetic acid (TFA) and the collagen peptides extracted using 100 μL C18 resin ZipTip® pipette tips (EMD Millipore). Samples were spotted in triplicate, along with calibration standards, onto a Bruker ground steel target plate using 1 μL each of extracted collagen and matrix solution (α-cyano-hydroxycinnamic acid). MALDI-ToF-MS was performed on the samples using a Bruker Ultraflex III mass spectrometer. Spectra were analyzed using mMass software^{48 (link)} and the resultant averaged spectrum for each sample identified by comparing them with published data^{19 (link),49 ,50 (link)}.

Each glycan sample (1 μL) was mixed on target with 1 μL matrix (5 μg/μL sDHB in 10% aqueous ACN). The mixture was allowed to dry at room temperature. MALDI-TOF-MS spectra were acquired in positive reflector mode on an Ultraflex III mass spectrometer (Bruker Daltonics, Bremen, Germany), equipped with a Smartbeam laser (337 nm) and a LIFT-MS/MS facility. Calibration was performed using a glucose ladder. In total, 5000 laser shots were accumulated for each mass spectrum. Spectra for free-glycan and O-glycan analyses were recorded in the mass range of 0–3000 Da, whereas a mass range of 1000–5000 Da was used for N-glycans. Collision-induced dissociation was performed using a collision energy of 29 kV. LIFT spectra consisted of at least 2500 laser shots. FlexAnalysis version 3.4 (Bruker Daltonics, Bremen, Germany) was used for baseline correction and peak picking. Glycan structures were annotated using GlycoPeakfinder [73 (link)] and assigned glycan structures were generated with the GlycoWorkbench software version 2.1 [74 (link),75 (link)]. Relative areas were determined for all glycan structures in all the samples.