Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy

Abstract

The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD-or ETD-based tandem mass spectrometry alone. (J Am Soc Mass Spectrom 2009, 20, 167-175)

Trypanosoma cruzi Bromodomain Factor 3 Binds Acetylated α-Tubulin and Concentrates in the Flagellum during Metacyclogenesis

“…Also, the TFPP server (a tool for recognizing flagellar proteins in T. brucei) (35) predicted a flagellar localization for TcBDF3 and its orthologue in T. brucei (Tb927.11.10070). Although Tb927.11.10070 has not been identified in the flagellar proteome of T. brucei (36)(37)(38), it is worth mentioning that, due to technical limitations, many flagellar proteins fail to be detected or cannot be assigned to the flagellum with certainty by proteomic studies.…”

Independent Analysis of the Flagellum Surface and Matrix Proteomes Provides Insight into Flagellum Signaling in Mammalian-infectious Trypanosoma brucei

“…Proteins with an expect-value of Յ1 ϫ 10 Ϫ10 were considered as homologs and identical criteria were applied across all data sets and comparisons. For T. brucei flagellum skeleton proteomes, we used the combined, nonoverlapping set of proteins identified in three proteomic analyses of extracted flagellum skeletons from procyclic-form parasites (31,43,46). Comparison to the C. reinhardtii flagellum proteome was done locally with proteins downloaded from (64).…”

“…Existing procedures employ detergent-extraction and salt-extraction to isolate the insoluble flagellum skeleton, which contains the axoneme and PFR, but lacks the membrane and matrix (43)(44)(45). Thus, although several hundred axonemal and PFR proteins have been identified (31,43,46), the protein compositions of the flagellar membrane and matrix in T. brucei are largely unknown. This poses a particular limitation for studying flagellum signaling, because signaling capacity is dictated by surface-exposed membrane proteins coupled to soluble components of signaling cascades in the matrix (2).…”

CMF70 is a subunit of the dynein regulatory complex

“…CMF70 is stably associated with the flagellum in T. brucei CMF70 was detected in one proteomic analysis of detergent and salt-extracted axonemes from T. brucei (Hart et al, 2009), although not in another (Broadhead et al, 2006). To further investigate CMF70, we used in situ tagging (Oberholzer et al, 2006) to replace one CMF70 allele with an epitope-tagged copy.…”