Shedding light on both ends: An update on analytical approaches for N- and C-terminomics

Koudelka, Tomas; Winkels, Konrad; Kaleja, Patrick; Tholey, Andreas

doi:10.1016/j.bbamcr.2021.119137

Cited by 11 publications

(10 citation statements)

References 102 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2 a). Such neo-N-termini can be identified using N-terminomics [ 35 ]. To this end, we performed negative enrichment via HYTANE [ 30 ] on serum-free conditioned media collected from SPPL3 KO and parental HEK293 cells to selectively capture neo-N-termini and to identify substrates of endogenous SPPL3.…”

Section: Resultsmentioning

confidence: 99%

N-terminome analyses underscore the prevalence of SPPL3-mediated intramembrane proteolysis among Golgi-resident enzymes and its role in Golgi enzyme secretion

Hobohm

Koudelka

Bahr

et al. 2022

Cell. Mol. Life Sci.

Self Cite

View full text Add to dashboard Cite

Golgi membrane proteins such as glycosyltransferases and other glycan-modifying enzymes are key to glycosylation of proteins and lipids. Secretion of soluble Golgi enzymes that are released from their membrane anchor by endoprotease activity is a wide-spread yet largely unexplored phenomenon. The intramembrane protease SPPL3 can specifically cleave select Golgi enzymes, enabling their secretion and concomitantly altering global cellular glycosylation, yet the entire range of Golgi enzymes cleaved by SPPL3 under physiological conditions remains to be defined. Here, we established isogenic SPPL3-deficient HEK293 and HeLa cell lines and applied N-terminomics to identify substrates cleaved by SPPL3 and released into cell culture supernatants. With high confidence, our study identifies more than 20 substrates of SPPL3, including entirely novel substrates. Notably, our N-terminome analyses provide a comprehensive list of SPPL3 cleavage sites demonstrating that SPPL3-mediated shedding of Golgi enzymes occurs through intramembrane proteolysis. Through the use of chimeric glycosyltransferase constructs we show that transmembrane domains can determine cleavage by SPPL3. Using our cleavage site data, we surveyed public proteome data and found that SPPL3 cleavage products are present in human blood. We also generated HEK293 knock-in cells expressing the active site mutant D271A from the endogenous SPPL3 locus. Immunoblot analyses revealed that secretion of select novel substrates such as the key mucin-type O-glycosylation enzyme GALNT2 is dependent on endogenous SPPL3 protease activity. In sum, our study expands the spectrum of known physiological substrates of SPPL3 corroborating its significant role in Golgi enzyme turnover and secretion as well as in the regulation of global glycosylation pathways.

show abstract

Section: Resultsmentioning

confidence: 99%

N-terminome analyses underscore the prevalence of SPPL3-mediated intramembrane proteolysis among Golgi-resident enzymes and its role in Golgi enzyme secretion

Hobohm

Koudelka

Bahr

et al. 2022

Cell. Mol. Life Sci.

Self Cite

View full text Add to dashboard Cite

show abstract

“…This can be caused by processes such as RNA-splicing or DNA-recombination as well as by a plethora of co- and post-translational modifications, for example, acylation, pyroglutamate formation at N-terminal glutamate residues, and amidations. − A major mechanism for the formation of non-encoded N- and C-termini is proteolytic processing. The composition of protein N- and C-termini has a strong influence on the physicochemical properties of proteins and hence directly on their biological function. , Therefore, the proper identification of protein termini by means of terminomics approaches, which are mainly based on liquid chromatography–mass spectrometry (LC–MS), is a major task. , …”

Section: Introductionmentioning

confidence: 99%

“…With a few exceptions, , these peptide-centric approaches do not target both termini in a single experiment. − Here lies one major advantage of TDP, which provides information on both termini in a single experiment without losing the connection between the respective N- and C-termini of a given proteoform. However, due to the above-mentioned technical challenges, only a limited number of publications have employed TDP for a directed investigation of protein termini, while its potential for terminomics has been described several times. ,− Most other TDP studies report results derived from subsequence searches but seldom focus on the interpretation of N- and C-terminal truncations.…”

Section: Introductionmentioning

confidence: 99%

“…4,5 Therefore, the proper identification of protein termini by means of terminomics approaches, which are mainly based on liquid chromatography−mass spectrometry (LC−MS), is a major task. 5,6 Proteomics experiments can be performed using either bottom-up proteomics (BUP) or top-down proteomics (TDP) approaches. In BUP, proteins are cleaved by the use of specific proteases to generate peptides.…”

Section: ■ Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Validation of Top-Down Proteomics Data by Bottom-Up-Based N-Terminomics Reveals Pitfalls in Top-Down-Based Terminomics Workflows

et al. 2022

Self Cite

View full text Add to dashboard Cite

Bottom-up proteomics (BUP)-based N-terminomics techniques have become standard to identify protein N-termini. While these methods rely on the identification of N-terminal peptides only, top-down proteomics (TDP) comes with the promise to provide additional information about post-translational modifications and the respective C-termini. To evaluate the potential of TDP for terminomics, two established TDP workflows were employed for the proteome analysis of the nematode Caenorhabditis elegans. The N-termini of the identified proteoforms were validated using a BUP-based N-terminomics approach. The TDP workflows used here identified 1658 proteoforms, the N-termini of which were verified by BUP in 25% of entities only. Caveats in both the BUP- and TDP-based workflows were shown to contribute to this low overlap. In BUP, the use of trypsin prohibits the detection of arginine-rich or arginine-deficient N-termini, while in TDP, the formation of artificially generated termini was observed in particular in a workflow encompassing sample treatment with high acid concentrations. Furthermore, we demonstrate the applicability of reductive dimethylation in TDP to confirm biological N-termini. Overall, our study shows not only the potential but also current limitations of TDP for terminomics studies and also presents suggestions for future developments, for example, for data quality control, allowing improvement of the detection of protein termini by TDP.

show abstract

“…Proteomics technologies have emerged as a powerful tool for the systematical and high-throughput analysis of protein termini. Thus far, various strategies have been developed for protein termini enrichment ( Huesgen and Overall, 2012 ; Rogers and Overall, 2013 ; Koudelka et al, 2021 ). However, current methods for protein C-terminome profiling still lag far behind the N-terminomics technologies.…”

Section: Introductionmentioning

confidence: 99%

PBC, an easy and efficient strategy for high-throughput protein C-terminome profiling

Zhai

Wang

et al. 2022

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

High-throughput profiling of protein C-termini is still a challenging task. Proteomics provides a powerful technology for systematic and high-throughput study of protein C-termini. Various C-terminal peptide enrichment strategies based on chemical derivatization and chromatography separation have been reported. However, they are still costly and time-consuming, with low enrichment efficiency for C-terminal peptides. In this study, by taking advantage of the high reaction selectivity of 2-pyridinecarboxaldehyde (2-PCA) with an α-amino group on peptide N-terminus and high affinity between biotin and streptavidin, we developed a 2-PCA- and biotin labeling–based C-terminomic (PBC) strategy for a high-efficiency and high-throughput analysis of protein C-terminome. Triplicates of PBC experiments identified a total of 1,975 C-terminal peptides corresponding to 1,190 proteins from 293 T cell line, which is 180% higher than the highest reported number of C-terminal peptides identified from mammalian cells by chemical derivatization–based C-terminomics study. The enrichment efficiency (68%) is the highest among the C-terminomics methods currently reported. In addition, we not only uncovered 50 proteins with truncated C-termini which were significantly enriched in extracellular exosome, vesicle, and ribosome by a bioinformatic analysis but also systematically characterized the whole PTMs on C-terminal in 293 T cells, suggesting PBC as a powerful tool for protein C-terminal degradomics and PTMs investigation. In conclusion, the PBC strategy would benefit high-efficiency and high-throughput profiling of protein C-terminome.

show abstract

Shedding light on both ends: An update on analytical approaches for N- and C-terminomics

Cited by 11 publications

References 102 publications

N-terminome analyses underscore the prevalence of SPPL3-mediated intramembrane proteolysis among Golgi-resident enzymes and its role in Golgi enzyme secretion

N-terminome analyses underscore the prevalence of SPPL3-mediated intramembrane proteolysis among Golgi-resident enzymes and its role in Golgi enzyme secretion

Validation of Top-Down Proteomics Data by Bottom-Up-Based N-Terminomics Reveals Pitfalls in Top-Down-Based Terminomics Workflows

PBC, an easy and efficient strategy for high-throughput protein C-terminome profiling

Contact Info

Product

Resources

About