High-Resolution Ion-Mobility-Enabled Peptide Mapping for High-Throughput Critical Quality Attribute Monitoring.

Characterization and monitoring of post-translational modifications (PTMs) by peptide mapping is a ubiquitous assay in biopharmaceutical characterization. Often, this assay is coupled to reversed-phase liquid chromatographic (LC) separations that require long gradients to identify all components of the protein digest and resolve critical modifications for relative quantitation. Incorporating ion mobility (IM) as an orthogonal separation that relies on peptide structure can supplement the LC separation by providing an additional differentiation filter to resolve isobaric peptides, potentially reducing ambiguity in identification through mobility-aligned fragmentation and helping to reduce the run time of peptide mapping assays.

Nucleation Inhibition of Huntingtin Protein (htt) by Polyproline PPII Helices: A Potential Interaction with the N-Terminal α-Helical Region of Htt.

Huntington's disease is a genetic neurodegenerative disorder characterized by the formation of amyloid fibrils of the huntingtin protein (htt). The 17-residue N-terminal region of htt (Nt) has been implicated in the formation of early phase oligomeric species, which may be neurotoxic. Because tertiary interactions with a downstream (C-terminal) polyproline (polyP) region of htt may disrupt the formation of oligomers, which are precursors to fibrillar species, the effect of co-incubation of a region of htt with a 10-residue polyP peptide on oligomerization and fibrillization has been examined by atomic force microscopy.

Acetylation within the First 17 Residues of Huntingtin Exon 1 Alters Aggregation and Lipid Binding.

Huntington's disease (HD) is a genetic neurodegenerative disorder caused by an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ leads to htt aggregation. The first 17 amino acids (Nt(17)) in htt comprise a lipid-binding domain that undergoes a number of posttranslational modifications that can modulate htt toxicity and subcellular localization.

Huntingtin N-Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues.

Early stage oligomer formation of the huntingtin protein may be driven by self-association of the 17-residue amphipathic α-helix at the protein's N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington's disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation.

Online deuterium hydrogen exchange and protein digestion coupled with ion mobility spectrometry and tandem mass spectrometry.

Online deuterium hydrogen exchange (DHX) and pepsin digestion (PD) is demonstrated using drift tube ion mobility spectrometry (DTIMS) coupled with linear ion trap (LTQ) mass spectrometry (MS) with electron transfer dissociation (ETD) capabilities. DHX of deuterated ubiquitin, followed by subsequent quenching and digestion, is performed within ∼60 s, yielding 100% peptide sequence coverage. The high reproducibility of the IMS separation allows spectral feature matching between two-dimensional IMS-MS datasets (undeuterated and deuterated) without the need for dataset alignment.

The emerging role of the first 17 amino acids of huntingtin in Huntington's disease.

Huntington's disease (HD) is caused by a polyglutamine (polyQ) domain that is expanded beyond a critical threshold near the N-terminus of the huntingtin (htt) protein, directly leading to htt aggregation. While full-length htt is a large (on the order of ∼350 kDa) protein, it is proteolyzed into a variety of N-terminal fragments that accumulate in oligomers, fibrils, and larger aggregates. It is clear that polyQ length is a key determinant of htt aggregation and toxicity.

Lysine residues in the N-terminal huntingtin amphipathic α-helix play a key role in peptide aggregation.

Huntington's disease is a genetic neurodegenerative disorder caused by an expansion in a polyglutamine domain near the N-terminus of the huntingtin (htt) protein that results in the formation of protein aggregates. Here, htt aggregate structure has been examined using hydrogen-deuterium exchange techniques coupled with tandem mass spectrometry. The focus of the study is on the 17-residue N-terminal flanking region of the peptide that has been shown to alter htt aggregation kinetics and morphology.

Combining ion mobility spectrometry with hydrogen-deuterium exchange and top-down MS for peptide ion structure analysis.

The gas-phase conformations of electrosprayed ions of the model peptide KKDDDDIIKIIK have been examined by ion mobility spectrometry (IMS) and hydrogen deuterium exchange (HDX)-tandem mass spectrometry (MS/MS) techniques. [M+4H](4+) ions exhibit two conformers with collision cross sections of 418 Å(2) and 471 Å(2). [M+3H](3+) ions exhibit a predominant conformer with a collision cross section of 340 Å(2) as well as an unresolved conformer (shoulder) with a collision cross section of ~367 Å(2).

A new ion mobility-linear ion trap instrument for complex mixture analysis.

A new instrument that couples a low-pressure drift tube with a linear ion trap mass spectrometer is demonstrated for complex mixture analysis. The combination of the low-pressure separation with the ion trapping capabilities provides several benefits for complex mixture analysis. These include high sensitivity, unique ion fragmentation capabilities, and high reproducibility.