Alexandra van Remoortere1, René J. M. van Zeijl1, Nico van den Oever2, Julien Franck3, Rémi Longuespée3, Maxence Wisztorski3, Michel Salzet3, André M. Deelder1, Isabelle Fournier3, and Liam A. McDonnell1
- Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands
- Hogeschool Leiden, Leiden, The Netherlands
- Université de Lille1, CNRS-FRE 3249, MALDI Imaging Team, Laboratoire de Neuroimmunologie et Neurochimie Evolutives, Villeneuve d’Ascq, France
MALDI imaging and profiling mass spectrometry of proteins typically leads to the detection of a large number of peptides and small proteins but is much less successful for larger proteins: most ion signals correspond to proteins of m/z < 25,000. This is a severe limitation as many proteins, including cytokines, growth factors, enzymes, and receptors have molecular weights exceeding 25 kDa. The detector technology typically used for protein imaging, a microchannel plate, is not well suited to the detection of high m/z ions and is prone to detector saturation when analyzing complex mixtures. Here we report increased sensitivity for higher mass proteins by using the CovalX high mass HM1 detector (Zurich, Switzerland), which has been specifically designed for the detection of high mass ions and which is much less prone to detector saturation. The results demonstrate that a range of different sample preparation strategies enable higher mass proteins to be analyzed if the detector technology maintains high detection efficiency throughout the mass range. The detector enables proteins up to 70 kDa to be imaged, and proteins up to 110 kDa to be detected, directly from tissue, and indicates new directions by which the mass range amenable to MALDI imaging MS and MALDI profiling MS may be extended.
CovalX Technology Used (Click each option to learn more)
CovalX has designed a detection system specifically for higher mass proteins and protein complexes. The HM1 and HM2 use an ion conversion detector which greatly increases the sensitivity of the mass spectrometer. Higher mass ions: incident ions collide with a conversion dynode array which creates smaller secondary ions. Then, the secondary ions are reaccelerated into a SEM and detected with higher sensitivity because of their higher velocity. The use of these detector systems for high mass protein ions helps to prevent detector saturation and allow for better sensitivity and profiling.
Experiments were performed by obtaining 12 μM thick tissue samples from the brains of adult male Wistar rats. The tissues were mounted onto conductive glass slides, frozen for an hour and washed. Following washing, the tissue sections were placed in a bath of cold acetone for 30 seconds, removed and a bath of cold 95% ethanol for another 30 seconds and finally, placed in chloroform for 1 minute. Samples were compared using different procedures (standard, HFIP, Leinweber, tween and H2O2) before 0.5 μL of the specific matrix was added using a micropipette or an automated sprayer. Each sample preparation was analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.
Standard sample preparation: chloroform and ethanol washes, mixed with matrix of 20 mg/ml sinapic acid in acetonitrile:0.1% TFA (&;3, v/v)
HFIP sample preparation: addition of 20 mg/ml sinapic acid in pure HFIP onto washed tissue and then addition of recrystallization solution (20 mg/mL sinapic acid in acetonitrile:0.1% TFA (7:3, v/v))
Leinweber sample preparation: placement of tissue samples onto a droplet of 20 mg/ml sinapic acid in 90% ethanol that contained 0.5 % Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether and 0.1% TFA. The droplet was allowed to dry and a droplet of sonicated sinapic acid suspended in xylene was placed on top of the tissue sample before being dried in a vacuum desiccator. Finally, matrix droplets were applied again using 20 mg/mL sinapic acid solutions in 90% ethanol and 50% acetonitrile.
Tween sample preparation: the tissues were washed and a matrix solution (20 mg/ml sinapic acid in acetonitrile:0.1% TFA with a low concentration of polyethylene glycol sorbitan monolaurate) was added and allowed to dry.
H2O2 sample preparation: tissues were covered in a 3% solution of H2O2 before being incubated in a saturated vapor pressure chamber for 30 minutes and then dried in a vacuum desiccator. Finally, the samples were prepared using a matrix solution (20 mg/ml sinapic acid in acetonitrile:0.1% TFA (7:3, v/v)).