Yuko P. Y. Lam

Two-dimensional mass spectrometry (2DMS) is a truly data-independent acquisition technique used in the analysis of complex mixtures; however, the nature of the noise within these spectra is not well understood. In this work, 2DMS is tested for conformity with the Fellgett principle: (signal/noise) proportional to root (no. of data points). Since 2DMS functions through the modulation of ions through a fragmentation region across many scans, the individual scans are considered data points in this experiment. Random noise was shown to be prevalent as the main source of noise in this experiment with minor systematic noise. This means that the minimum size for a 2DMS spectrum that displays a target fragment ion can be determined using a fast-2D equation detailed herein. The effects of existing denoising algorithms were also found to change the relationship between the signal-to-noise ratio and the scan numbers to be of a quasi-linear nature rather than the square root trend observed before denoising.

Multimodal tandem mass spectrometry sequencing of oligonucleotides using CID, EDD, IRMPD, and UVPD as fragmentation methods, and using 2-dimensional mass spectrometry with signal averaging to differentiate fragments from closely spaced precursors.

We demonstrate that the Parkinson’s VPS35[D620N] mutation alters the expression of ~220 lysosomal proteins and stimulates recruitment and phosphorylation of Rab proteins at the lysosome. This recruits the phospho-Rab effector protein RILPL1 to the lysosome where it binds to the lysosomal integral membrane protein TMEM55B. We identify highly conserved regions of RILPL1 and TMEM55B that interact and design mutations that block binding. In mouse fibroblasts, brain, and lung, we demonstrate that the VPS35[D620N] mutation reduces RILPL1 levels, in a manner reversed by LRRK2 inhibition and proteasome inhibitors. Knockout of RILPL1 enhances phosphorylation of Rab substrates, and knockout of TMEM55B increases RILPL1 levels. The lysosomotropic agent LLOMe also induced LRRK2 kinase–mediated association of RILPL1 to the lysosome, but to a lower extent than the D620N mutation. Our study uncovers a pathway through which dysfunctional lysosomes resulting from the VPS35[D620N] mutation recruit and activate LRRK2 on the lysosomal surface, driving assembly of the RILPL1-TMEM55B complex.

A diagram to show the differences between wild-type and deamidated hIAPPs interaction with site specific and non-specific inhibitors.

The Os^II arene anticancer complex [(η⁶-bip)Os(en)Cl]⁺ (Os1–Cl; where bip = biphenyl and en = ethylenediamine) binds strongly to DNA¹ and biomolecules. Here we investigate the interaction between Os1–Cl and the model protein, BSA, using ultrahigh resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS). The specific binding location of Os1 on BSA was investigated with the use of collisionally activated dissociation (CAD) and electron capture dissociation (ECD). CAD MS/MS was found to dissociate the osmium complex from the metallo–peptide complex readily producing unmodified fragments and losing location information. ECD MS/MS, however, successfully retains the osmium modification on the peptides upon fragmentation allowing localization of metallocomplex binding. This study reveals that lysine is a possible binding location for Os1–Cl, apart from the expected binding sites at methionine, histidine, and cysteine. Using a nano liquid chromatography (nLC)–FT-ICR ECD MS/MS study, multiple binding locations, including the N-terminus and C-terminus of digested peptides, glutamic acid, and lysine were also revealed. These results show the multitargeting binding ability of the organo-osmium compound and can be used as a standard workflow for more complex systems, e.g., metallocomplex–cell MS analysis, to evaluate their behavior toward commonly encountered biomolecules.

Amyloid fibril formation is a hallmark in a range of human diseases. Analysis of the molecular details of amyloid aggregation, however, is limited by the difficulties in solubilizing, separating, and identifying the aggregated biomolecules. Additional labeling or protein modification is required in many current analytical techniques in order to provide molecular details of amyloid protein aggregation, but these modifications may result in protein structure disruption. Herein, ultrahigh resolution mass spectrometry (MS) with electron capture dissociation tandem MS (ECD MS/MS) has been applied to monitor the formation of early oligomers of human islet amyloid polypeptide (hIAPP), which aggregate rapidly in the pancreas of type II diabetes (T2D) patients. ECD MS/MS results show the aggregation region of the early oligomers is at the Ser-28/Ser-29 residue of a hIAPP unit and at the Asn-35 residue of another hIAPP unit near the C-terminus in the gas phase. These data contribute to the understanding of the binding site between hIAPP units which may help for specific target region therapeutic development in the future. Furthermore, MS has also been applied to quantify the amount of soluble amyloid protein remaining in the incubated solutions, which can be used to estimate the aggregation rate of amyloid protein during incubation (28 days). These data are further correlated with the results obtained using fluorescence spectroscopy and transmission electron microscopy (TEM) to generate a general overview of amyloid protein aggregation. The methods demonstrated in this article not only explore the aggregation site of hIAPP down to an amino acid residue level, but are also applicable to many amyloid protein aggregation studies.

Native top-down mass spectrometry is a fast, robust biophysical technique that can provide molecular-scale information on the interaction between proteins or peptides and ligands, including metal c...

Fourier transform ion cyclotron resonance mass analysers (FT-ICR MS) can offer the highest resolutions and mass accuracies in mass spectrometry. Mass spectra acquired in an FT-ICR MS can yield accurate elemental compositions of all compounds in a complex sample. Fragmentation caused by ion–neutral, ion–electron, or ion–photon interactions leads to more detailed structural information on compounds. The most often used method to correlate compounds and their fragment ions is to isolate the precursor ions from the sample before fragmentation. Two-dimensional mass spectrometry (2D MS) offers a method to correlate precursor and fragment ions without requiring precursor isolation. 2D MS therefore enables easy access to the fragmentation patterns of all compounds from complex samples. In this article, the principles of FT-ICR MS are reviewed and the 2D MS experiment is explained. Data processing for 2D MS is detailed, and the interpretation of 2D mass spectra is described.

Deamidation sites of hIAPP were determined and the amount of fibril formed in mutant hIAPPs was higher than in wild-type hIAPP.

Two-dimensional mass spectrometry (2D MS) correlates precursor and fragment ions without ion isolation in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for tandem mass spectrometry. Infrared activated electron capture dissociation (IR-ECD), using a hollow cathode configuration, generally yields more information for peptide sequencing in tandem mass spectrometry than ECD (electron capture dissociation) alone. The effects of the fragmentation zone on the 2D mass spectrum are investigated, as well as the structural information that can be derived from it. The enhanced structural information gathered from the 2D mass spectrum is discussed in terms of how de novo peptide sequencing can be performed with increased confidence. 2D IR-ECD MS is shown to sequence peptides, produces w ions which enable the distinction between leucine and isoleucine residues, C-terminal and N-terminal fragments, as well as the assignment and location of peptide modifications.