M5 sprayer

Serum sample preparation was performed as previously described^{34 (link)}. Serum samples were diluted in sodium bicarbonate and spotted onto an amine-reactive slide. Each slide had spot of PBS diluted in sodium bicarbonate added as a blank. Additionally, a standard healthy serum sample was added to each slide for normalization across slides. All samples and standards were spotted in technical triplicate. The slide was then placed into a humidity chamber for 1 h to bind the serum proteins to the slide. Well chambers were attached to the slide to isolate each sample, and the samples were washed with Carnoy’s solution and water to remove lipids and salts, respectively. After drying the slides, PNGase F was sprayed across the slide with an automated sprayer (M5 TM-Sprayer, HTX Technologies, Chapel Hill, NC) and incubated in a humidity chamber at 37 °C for 2 h to enzymatically cleave the N-glycans from the captured serum glycoproteins. The HTX M5 sprayer was then used to spray CHCA matrix across the slide.

A standardized protocol was used for tissue preparation, enzymatic digestion, and matrix application by a solvent sprayer (9 ). Tissue slides were dewaxed in xylenes and then rehydrated in a gradation of EtOH and water washes before antigen retrieval with citraconic anhydride buffer (25-μl citraconic anhydride, 2-μl 12 M HCl, 50-ml HPLC-grade water, pH 3.0 ± 0.5) for 30 min in a decloaking chamber at 95 °C. Fifteen passes of PNGase F or 10% Endo F3/90% PNGase F at 0.1 μg/μl was then sprayed onto the slides at a rate of 25 μl/min with a 3-mm offset and a velocity of 1200 at 45 °C and 10 psi using an M5 TM Sprayer (HTX Technologies LLC). Tissue slides were then incubated at 37 °C for 2 h in prewarmed humidity chambers followed by desiccation before matrix application. 10 passes of 7 mg/ml CHCA matrix in 50% acetonitrile/0.1% TFA was applied to the desiccated slides at 0.1 ml/min with a 2.5-mm offset and a velocity of 1300 at 80 °C and 10 psi using the TM Sprayer. After matrix application slides were desiccated until analysis by MALDI–Fourier-transform ion cyclotron resonance (FTICR) MS or MALDI quadrupole time-of-flight mass spectrometry (MALDI–QTOF MS). Postanalysis whole-tissue slides were H&E-stained according to a standardized protocol and annotated for histological classification by an expert pathologist.

N-glycan profiling by MALDI-MSI was performed using a standardized protocol, as previously described (Powers et al., 2014 (link); Drake et al., 2018 (link)). FFPE tissues were dewaxed by heating at 60°C for 1 h, washed in xylene, and rehydrated using a graded series of ethanol and water. Slides were antigen retrieved in citraconic anhydride buffer (25 µL citraconic anhydride, 2 µL 12 M HCl, 50 mL of HPLC water, pH 3.0 ± 0.5) for 20 min in a decloaking chamber at 95°C. Fifteen passes of 0.1 μg/μL PNGase F PRIME were applied using an M5 TM Sprayer (25 μL/min flow rate, 1,200 mm/min, 45°C, 3 mm offset, 10 psi nitrogen gas, HTX Technologies LC). Slides were incubated for 2 h at 37°C in preheated humidified chambers. After desiccation, 7 mg/mL CHCA matrix in 50% acetonitrile/0.1% TFA was applied using a TM Sprayer (10 passes, 100 μL/min flow rate, 1,300 mm/min, 79°C, 2.5 mm offset, 10 psi nitrogen). The slides were stored in a desiccator until analysis.

Samples were vacuum desiccated for 30 min and then washed in fresh solutions of 70% ethanol for 30 s, 100% ethanol for 30 s, Carnoy’s solution (6:3:1 v/v ethanol/chloroform/glacial acetic acid) for 2 min, 100% ethanol for 30 s, water with 0.2% TFA for 15 s, and 100% ethanol for 30 s. Samples were then dried by a stream of nitrogen gas prior to MALDI matrix application. HTX Technologies M5 Sprayer was used to deposit sonicated supernatant of 15 mg/ml 2,5-DHA (2,5-dihydroxyacetophenone) in 90% acetonitrile with 0.2% TFA. The flow rate of the matrix was 150 μl/min with a nozzle temperature of 30.0 °C, with a velocity set to 1300 mm/min with 10 PSI of nitrogen gas. The matrix was then recrystallized with 5% acetic acid solution in water at 38.5 °C and dried for 3.5 min and then immediately analyzed using an elevated pressure MALDI source (Spectroglyph LLC) coupled to a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with ultra-high mass range boards (28 (link)). Spectra were acquired over the m/z range of 3500 to 20,000 in positive polarity mode with a resolving power of 240k at m/z 200 (512 ms transient) and 250 laser shots per pixel. Scans in the.RAW file were summed as a single spectrum for proteoform assignment by accurate mass.