Best practices and benchmarks for intact protein analysis for top-down mass spectrometry

Abstract

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

Quantifying the heterogeneity of macromolecular machines by mass photometry

“…Given that different oligomeric complexes vary in molecular mass, mass measurement could in principle be ideally suited to examine sample heterogeneity. Despite the advances in native mass spectrometry (MS) over the past decades [13][14][15] , the associated experimental complexity and non-native conditions have prevented native MS from becoming a widely used tool in this context. Mass photometry (MP), originally introduced as interferometric scattering mass spectrometry (iSCAMS), is a label-free approach that accurately measures molecular mass by quantifying light scattering from single biomolecules in solution 16,17 .…”

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

“…The review of Schaffer et al is recommended as an introduction into TDMS [9]. Robust protocols for mass analysis of intact proteins with TDMS were recently published by Donnelly et al [10]. TDMS is requiring sample mixtures of low complexity for obtaining high quality spectra of proteoforms.…”

FLASHDeconv: Ultrafast, high-quality feature deconvolution for top-down proteomics

“…Let mi be the maximum number of intense charges that continuously increment by i. For example, if charges 3, 5, 6, 7, 9, 10 are intense, m1 = 3 (charges 5, 6, 7) while m2 = 4 (charges 3,5,7,9). A peak group is filtered out if m1 < max (3, m2,m3,...,m7).…”

“…Top-down (TD) proteomics has gained a lot of momentum for in-depth protein characterization and protein species analytics [1][2][3][4][5][6][7] . In contrast to bottom-up (BU) proteomics, where proteins are enzymatically digested and actually peptides are analyzed, the TD approach allows for the analysis of intact proteoforms (protein species arising from the same gene product via splice variants, genomic variation, post-translational modifications, degradation, etc.)…”

“…Current MS instrumentation and experimental protocols enable more complex analyses using TD proteomics, but in turn yield equally complex and large data sets 1,2,6,7,10,12 . Hence, both quality and runtime of deconvolution algorithms have become an issue.…”