Reliable determination of protein complex composition or changes to protein levels in whole cells is challenging. Despite the multitude of methods now available for labeling, analysis, and the statistical processing of data, this large variety is of itself an issue: Which approach is most appropriate, where do you set cutoffs, and what is the most cost-effective strategy? One size does not fit all for such work, but some guidelines can help in terms of reducing cost, improving data quality, and ultimately advancing investigations. Here we describe two protocols and algorithms for facile sample preparation for mass spectrometric analysis, robust data processing, and considerations of how to interpret large proteomic datasets in a productive and robust manner.

BIOCEV Department of Parasitology Faculty of Science Charles University Prague Vestec Czechia

Biology Centre Institute of Parasitology Faculty of Sciences University of South Bohemia České Budějovice Czechia

School of Life Sciences University of Dundee Dundee UK

Zobrazit více v PubMed

Ahmad Y, Lamond AI (2014) A perspective on proteomics in cell biology. Trends Cell Biol 24(4):257–264 PubMed DOI PMC

Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, Depledge DP, Fischer S, Gajria B, Gao X, Gardner MJ, Gingle A, Grant G, Harb OS, Heiges M, Hertz-Fowler C, Houston R, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Logan FJ, Miller JA, Mitra S, Myler PJ, Nayak V, Pennington C, Phan I, Pinney DF, Ramasamy G, Rogers MB, Roos DS, Ross C, Sivam D, Smith DF, Srinivasamoorthy G, Stoeckert CJ Jr, Subramanian S, Thibodeau R, Tivey A, Treatman C, Velarde G, Wang H (2010) TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res 38:D457–D462 PubMed DOI

Urbaniak MD, Guther ML, Ferguson MA (2012) Comparative SILAC proteomic analysis of Trypanosoma brucei bloodstream and procyclic lifecycle stages. PLoS One 7(5):e36619 PubMed DOI PMC

Matthews KR (2005) The developmental cell biology of Trypanosoma brucei. J Cell Sci 118:283–290 PubMed DOI

Makarov A (2000) Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis. Anal Chem 72:1156–1162 PubMed DOI

Keilhauer EC, Hein MY, Mann M (2015) Accurate protein complex retrieval by affinity enrichment mass spectrometry (AE-MS) rather than affinity purification mass spectrometry (AP-MS). Mol Cell Proteomics 14(1):120–135 PubMed DOI

Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13:2513–2526 PubMed DOI PMC

Obado SO, Field MC, Chait BT, Rout MP (2016) High-efficiency isolation of nuclear envelope protein complexes from trypanosomes. Methods Mol Biol 1411:67–80 PubMed DOI

Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372 PubMed DOI

Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13(9):731–740 PubMed DOI

Zoltner M, Krienitz N, Field MC, Kramer S (2018) Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA. PLoS Negl Trop Dis 12(7):e0006679 PubMed DOI PMC

Trypanosomes lack a canonical EJC but possess an UPF1 dependent NMD-like pathway

Detailed characterisation of the trypanosome nuclear pore architecture reveals conserved asymmetrical functional hubs that drive mRNA export

A unique mRNA decapping complex in trypanosomes

TUSK: a ubiquitin hydrolase complex modulating surface protein abundance in trypanosomes

Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared