Effect of dynamic exclusion and the use of FAIMS, DIA and MALDI-mass spectrometry imaging with ion mobility on amyloid protein identification

Abstract

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC-MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC-MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot's Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

Keywords: Amyloidosis; DDA; DIA; FAIMS; Laser capture microdissection; Light chain amyloidosis; MALDI-MSI; Transthyretin amyloidosis.

Fig. 1

Schematic diagram of the workflow for amyloidosis analysis using a LMD/LC–MS/MS b MALDI-MSI

Fig. 2

A Amyloidosis sub-types from tissue specimens analyzed by LMD/LC–MS/MS. B Total spectral counts for transthyretin protein from CR (+) ATTR specimens analyzed by ± FAIMS with no dynamic exclusion (DE). C Total number of proteins identified from CR (+) ATTR specimens with and without FAIMS with no dynamic exclusion (DE). D Total number of proteins identified with and without FAIMS (± FAIMS) with and without dynamic exclusion E Total spectral counts of amyloid proteins with and without FAIMS with and without dynamic exclusion (± DE)

Fig. 3

Two label free quantitation methods based on A Spectral counts based on peptide-spectral matches at the MS2 level versus B Extracted ion chromatogram (XIC) of peptide GSPAINVAVHVFR (m/z 683.88, + 2) at the MS1 level. C, D Volcano plots of Congo red negative heart biopsy (CRN-H-2) vs Congo red positive heart biopsy (ATTR-H-4) based on C total spectral counts versus D normalized abundances of all the proteins identified. E. Transthyretin total spectral counts versus F raw abundances of transthyretin from Congo red negative heart biopsy (CRN-H-2) versus Congo red positive heart biopsy (ATTR-H-4)

Fig. 4

A Mean total number of proteins identified from Congo red positive CR (+) and negative CR (−) samples analyzed by DDA versus DIA. B Total number of proteins identified from each replicate from each group (shown in Panel A). C Transthyretin protein raw abundances from CR (+) and CR (−) samples by DDA versus DIA. D Normalized abundances of the transthyretin protein from CR (+) and CR (−) specimens by DDA versus DIA. E Violin plot of % coefficient of variation from DDA versus DIA analysis of CR (+) versus CR (−) samples. F Venn diagram of significant proteins identified from DDA and DIA analysis of CR (+) and CR (−) samples. Volcano plots of proteins identified from CR(+) samples versus CR (−) samples analyzed by: G DDA with transthyretin protein as one of the most significantly enriched (p-value = 7.62) among the 31 significant proteins in the CR (+) samples (with p-values > 4.32) versus H DIA with transthyretin protein as one of the most significantly enriched (p-value = 8.33) among the 51 significant proteins identified in the CR (+) samples (with p-values > 4.32)

Fig. 5

A A tree map of biological processes enriched in the Congo red positive specimens B Venn diagram of common proteins from heart (ATTR-H-12); heart (ATTR-H-8); stomach (ATTR-S-11); heart (ATTR-H-9); C Protein interaction network by STRING and Cytoscape; D Gene ontology of the top biological processes

Fig. 6

A MALDI-MSI analysis of transthyretin peptide (ATTR) GSPAINVAVHVFR (m/z 1366.7) on FFPE heart tissue section from autopsy and biopsy samples using the Ultraflextreme mass spectrometer. Presence of serum amyloid P component peptides VGEYSLYIGR (m/z 1156.6) and ERVGEYSLYIGR (m/z 1441.9) were also mapped on the same tissue sections. B Mass mobility resolved interfering ion observed from the Congo red positive peptide extract spiked with the peptide digests of the transthyretin standard; C three overlapping peptides observed from complex FFPE heart tissue matrix resolved by TIMS; D peptide ion heat maps from transthyretin, serum amyloid P component and Apolipoprotein E; E segmentation analysis