2.2. MALDI Data Acquisition

MALDI imaging mass spectrometry experiments were performed using a Bruker Rapiflex Tissuetyper (Bruker Daltonics, Billerica, MA, USA) equipped with a Smartbeam 3D 10 kHz 355 nm Nd:YAG laser™ 3D laser. m/z calibration was accomplished via a linear external calibration using a mix of bovine insulin, equine cytochrome c, bovine ubiquitin I, and equine myoglobin (Bruker Daltonik, Billerica, MA, USA). Mass spectrometric analyses were performed in the linear, positive ionization mode over a mass-to-charge (m/z) range of m/z 1200 to m/z 20,000. The ionization laser was scanned across the tissue surface with a raster step size of 60 μm and spectra from 2000 laser shots per pixel (tissue position) were averaged to produce a spectrum. Low molecular weight crystallin fragments were also analyzed in a positive ion reflector MS mode, to improve mass resolution, over an m/z range of m/z 950 to m/z 5000 and with a laser raster step size of 150 µm. Spectra from 500 laser shots were used to produce an average spectrum. The mass spectrometer was calibrated with a peptide mixture (Leu-enkephalin, Angiotensin II, Fibrinopeptide B, ACTH fragment (1–24), and insulin chain B). Ion images were generated using FlexImaging software (Version 4.1, Bruker Daltonics, Billerica, MA, USA) using RMS normalization.

To measure the accurate masses of the signals observed in IMS data in the cortex and nucleus regions, tissue from the cortex (within 400 µm from the capsule) and inner nucleus region (within 500 µm from the center) of a 55 Y.O. (C_5_1) cataract lens and the cortex region of 54 Y.O. (N_5_1) normal transparent lens was captured by laser capture microdissection (LCM). The LCM procedure was conducted using a PALM UV Laser MicroBeam laser capture microdissection system (Carl-Zeiss, Oberkochen, Germany) on tissue sections of 12 µm thickness on glass slides. The capture was done using a laser pressure catapulting (LPC) method and 37% laser energy. An area of roughly 1 mm² was collected for each sample. The samples were collected in 20 µL of water and mixed with acetonitrile containing 2%TFA to final acetonitrile concentration of 5% and loaded on C18 SpinTips (Thermo Scientific, Rockford, IL, USA). The samples were washed with 0.1%TFA and eluted in 2.5 µL of 70% acetonitrile containing 0.1%TFA. The samples were mixed with 2.5 µL of saturated α-cyano-4-hydroxycinnamic acid (CHCA) matrix (Sigma-Aldrich, St Louis, MO, USA) in 70% acetonitrile (0.1%TFA) and masses were measured with high accuracy (<5 ppm) in a Bruker SolariX 15T FT-ICR mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with a Smartbeam II 2 kHz Nd:YAG (355 nm) laser. The mass spectrometer was externally calibrated prior to analysis using a peptide mixture (Leu-enkephalin, Angiotensin II, Fibrinopeptide B, ACTH fragment (1–24), and insulin chain B). The mass spectrometer was operated in positive ion mode and data were collected from m/z 700 to 4500 at a mass resolution of ~155,000 at m/z = 2360.2393.