Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer.

peer reviewe

MALDI mass spectrometry imaging of proteins exceeding 30 000 daltons

Background: Since its introduction 10 years ago by Caprioli and associates, MALDI mass spectrometry imaging has enabled spatial analysis of drugs, lipids, peptides, and polypeptides. In polypeptides, the detectable mass range is limited to small proteins with a mass less than 25 kDa. This is a limitation, as many proteins, including cytokines, growth factors, enzymes, and receptors have molecular weights, exceeding 25 kDa. In the present work, we report the development of a novel strategy to observe higher mass proteins up to 30 kDa. Material/Methods: We investigated the development of sample preparation methods based on hexafluoroisopropanol (1,1,1,3,3,3-hexaluoro-2-propanol) and 2,2,2-trifluoroethanol solvents for protein solubilization optimized for high-mass proteins. Results: We were, for the first time in mass spectrometry imaging, able to detect to proteins up to 70 kDa directly from tissue. These developments indicate future avenues by which the sensitivity of protein mass spectrometry imaging can be further improved. We applied these developments to ovarian cancer and demonstrate that protein are similar to that which can be obtained using 2D gel based analyses. Conclusions: Increasing the possibility of detecting proteins and high-mass proteins is key for developing direct tissue proteomics and especially any potential functional investigation. These data will open the door of a novel step in mass spectrometry imaging

MALDI mass spectrometry imaging of proteins exceeding 30 000 daltons

Background: Since its introduction 10 years ago by Caprioli and associates, MALDI mass spectrometry imaging has enabled spatial analysis of drugs, lipids, peptides, and polypeptides. In polypeptides, the detectable mass range is limited to small proteins with a mass less than 25 kDa. This is a limitation, as many proteins, including cytokines, growth factors, enzymes, and receptors have molecular weights, exceeding 25 kDa. In the present work, we report the development of a novel strategy to observe higher mass proteins up to 30 kDa. Material/Methods: We investigated the development of sample preparation methods based on hexafluoroisopropanol (1,1,1,3,3,3-hexaluoro-2-propanol) and 2,2,2-trifluoroethanol solvents for protein solubilization optimized for high-mass proteins. Results: We were, for the first time in mass spectrometry imaging, able to detect to proteins up to 70 kDa directly from tissue. These developments indicate future avenues by which the sensitivity of protein mass spectrometry imaging can be further improved. We applied these developments to ovarian cancer and demonstrate that protein are similar to that which can be obtained using 2D gel based analyses. Conclusions: Increasing the possibility of detecting proteins and high-mass proteins is key for developing direct tissue proteomics and especially any potential functional investigation. These data will open the door of a novel step in mass spectrometry imaging.Proteomic

MALDI Imaging and Profiling MS of Higher Mass Proteins from Tissue

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 CovaIX 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. (J Am Soc Mass Spectrom 2010, 21, 1922-1929) (C) 2010 American Society for Mass Spectrometr

Accelerated pre-senile systemic amyloidosis in PACAP knockout mice - a protective role of PACAP in age-related degenerative processes

Dysregulation of neuropeptides may play an important role in aging-induced impairments. Among them, pituitary adenylate cyclase activating polypeptide (PACAP) is a potent cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. We hypothesized that the progressive decline of PACAP throughout life, and the well-known general cytoprotective effects of PACAP lead to age-related pathophysiological changes in PACAP deficiency, supported by the increased vulnerability to various stressors of animals partially or totally lacking PACAP. Using young and aging CD1 PACAP knockout (KO) and wild type (WT) mice, we demonstrated pre-senile amyloidosis in young PACAP KO animals and showed that senile amyloidosis appeared accelerated, more generalized, more severe, and affected more individuals. Histopathology showed age-related systemic amyloidosis with mainly kidney, spleen, liver, skin, thyroid, intestinal, tracheal and esophageal involvement. Mass spectrometry-based proteomic analysis, re-confirmed with immunohistochemistry, revealed that apolipoprotein-AIV was the main amyloid protein in the deposits together with several accompanying proteins. Although the local amyloidogenic protein expression was disturbed in KO animals, no difference was found in laboratory lipid parameters, suggesting a complex pathway leading to increased age-related degeneration with amyloid deposit in the absence of PACAP. In spite of no marked inflammatory histological changes or blood test parameters, we detected a disturbed cytokine profile that possibly creates a pro-inflammatory milieu favoring amyloid deposition. In summary, here we describe accelerated systemic senile amyloidosis in PACAP gene deficient mice, which might indicate an early aging phenomenon in this mouse strain. Thus, PACAP KO mice could serve as a model of accelerated aging with human relevance. This article is protected by copyright. All rights reserved

MALDI Imaging and Profiling MS of Higher Mass Proteins from Tissue

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 CovaIX 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. (J Am Soc Mass Spectrom 2010, 21, 1922-1929) (C) 2010 American Society for Mass SpectrometryProteomic