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1.
Mol Cell Proteomics ; 18(4): 773-785, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30622160

RESUMEN

De novo peptide sequencing for large-scale proteomics remains challenging because of the lack of full coverage of ion series in tandem mass spectra. We developed a mirror protease of trypsin, acetylated LysargiNase (Ac-LysargiNase), with superior activity and stability. The mirror spectrum pairs derived from the Ac-LysargiNase and trypsin treated samples can generate full b and y ion series, which provide mutual complementarity of each other, and allow us to develop a novel algorithm, pNovoM, for de novo sequencing. Using pNovoM to sequence peptides of purified proteins, the accuracy of the sequence was close to 100%. More importantly, from a large-scale yeast proteome sample digested with trypsin and Ac-LysargiNase individually, 48% of all tandem mass spectra formed mirror spectrum pairs, 97% of which contained full coverage of ion series, resulting in precision de novo sequencing of full-length peptides by pNovoM. This enabled pNovoM to successfully sequence 21,249 peptides from 3,753 proteins and interpreted 44-152% more spectra than pNovo+ and PEAKS at a 5% FDR at the spectrum level. Moreover, the mirror protease strategy had an obvious advantage in sequencing long peptides. We believe that the combination of mirror protease strategy and pNovoM will be an effective approach for precision de novo sequencing on both single proteins and proteome samples.


Asunto(s)
Metaloproteasas/metabolismo , Péptidos/metabolismo , Proteómica/métodos , Análisis de Secuencia de Proteína/métodos , Tripsina/metabolismo , Acetilación , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Estabilidad de Enzimas , Péptidos/química , Proteoma/metabolismo
2.
Bioinformatics ; 31(20): 3249-53, 2015 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-26076724

RESUMEN

MOTIVATION: Proteogenomics has been well accepted as a tool to discover novel genes. In most conventional proteogenomic studies, a global false discovery rate is used to filter out false positives for identifying credible novel peptides. However, it has been found that the actual level of false positives in novel peptides is often out of control and behaves differently for different genomes. RESULTS: To quantitatively model this problem, we theoretically analyze the subgroup false discovery rates of annotated and novel peptides. Our analysis shows that the annotation completeness ratio of a genome is the dominant factor influencing the subgroup FDR of novel peptides. Experimental results on two real datasets of Escherichia coli and Mycobacterium tuberculosis support our conjecture. CONTACT: yfu@amss.ac.cn or xupingghy@gmail.com or smhe@ict.ac.cn SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Péptidos/química , Proteómica , Escherichia coli/genética , Anotación de Secuencia Molecular , Mycobacterium tuberculosis/genética
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