Naslov
New concepts in MS, MS/MS and MS/MS/MS peptide and protein MALDI de-novo sequencing

Autori
Cindrić, Mario ; Dodig, Ivana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
4th Mass Spectrometry in Biotechnology and Medicine (MSBM) Summer School, Dubrovnik, Croatia

Mjesto i datum
Dubrovnik, Hrvatska, 04.07.2010. - 10.07.2010

Vrsta sudjelovanja
Pozvano predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Mass spectrometry; Sequence; Amino acids; Derivatization

Sažetak
Unambiguous and complete analysis of amino acid sequence of a protein or a peptide should be the final result of the sequence analysis [1, 2]. Edman sequencing has proven to be a powerful and sensitive strategy for the amino acid sequence analysis. [1, 3] However, up to 80 % of all proteins naturally have blocked N-termini making them resistant to the Edman degradation reaction. On the other hand, in past 20 years protein and peptide sequencing has been more pronounced with mass spectrometry than with the classical Edman degradation for numerous reasons (e.g. better sensitivity and repeatability, ability of posttranslational modification analysis, ability of quantitative and high-throughput analyses) [3-5]. However, amino acid modifications and unfavorable charge distribution in peptides/proteins could aggravate the unambiguous analysis. Implementation of mobile and localized proton models and selective derivatization could bridge the differences between complete and partial sequence analysis. Even relatively small peptides (up to 5000 Da) could remain partially sequenced after MS or MS/MS analyses. Therefore, in recent years, multi-stage mass spectrometry (MS3 to MSn) has been introduced in order to elucidate the sequence or to confirm the data trustfulness [6-8]. Three new de novo sequencing concepts: Multiple Induced MALDI In-source Fragmentation (MIMIF), enhanced MALDI in-source decay (enhanced pseudo MS/MS and MS3) and negative/positive ion de novo sequencing will be elaborated in details. Negative/positive ion de novo sequencing is examined after long-term-incubation in nutrient wasted environment that affected on Lactobacillus brevis L62 gene expresion. The cells were grown for 75 days in spent MRS medium and 75-day-old isolates proteome was examined and compared with parent cells. Since Lactobacillus brevis proteome in not well known de novo sequencing is crucial for understanding metabolic processes in described model. [1] Klappera DG. Trends in automated protein sequence analysis. TrAC. 1983 Dec ; 2(12): 267-69. [2] Standing KG. Peptide and protein de novo sequencing by mass spectrometry. Curr. Opin. Struct. Biol. 2003 Oct ; 13(5): 595-01. [3] Patterson SD, Aebersold RH. Proteomics: the first decade and beyond... Nat Genet. 2003 Mar ; 33 Suppl:311-23. [4] Samgina TY, Kovalev SV, Gorshkov VA, Artemenko KA, Poljakov NB, Lebedev AT. N-terminal tagging strategy for de novo sequencing of short peptides by ESI-MS/MS and MALDI-MS/MS. J. Am. Soc. Mass Spectrom. 2010 Jan ; 21(1):104-11. [5] Deutzmann R. Structural characterization of proteins and peptides. Methods Mol. Med. 2004 Apr ; 94:269-97. [6] Yates JR 3rd, Morgan SF, Gatlin CL, Griffin PR, Eng JK. Method to compare collision-induced dissociation spectra of peptides: potential for library searching and subtractive analysis. Anal. Chem. 1998. Sep ; 70(17): 3557-65. [7] Han G, Ye M, Jiang X, Chen R, Ren J, Xue Y, Wang F, Song C, Yao X, Zou H. Comprehensive and reliable phosphorylation site mapping of individual phosphoproteins by combination of multiple stage mass spectrometric analysis with a target-decoy database search. Anal. Chem. 2009 Jul ; 81(14): 5794-5805. [8] Marzillia LA, Goldena TR, Cottera RJ, Woods AS. Peptide sequence information derived by pronase digestion and ammonium sulfate in-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J. Am. Soc. Mass Spectrom. 2000 Nov ; 11(11): 1000-08.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA