Top-down proteomics reveals novel protein forms expressed in Methanosarcina acetivorans - PubMed (original) (raw)

Top-down proteomics reveals novel protein forms expressed in Methanosarcina acetivorans

Jonathan T Ferguson et al. J Am Soc Mass Spectrom. 2009 Sep.

Abstract

Using both automated nanospray and online liquid chromatography mass spectrometry LC-MS strategies, 99 proteins have been newly identified by top-down tandem mass spectrometry (MS/MS) in Methanosarcina acetivorans, the methanogen with the largest known genome [5.7 mega base pairs (Mb)] for an Archaeon. Because top-down MS/MS was used, 15 proteins were detected with mispredicted start sites along with an additional five from small open reading frames (SORFs). Beyond characterization of these more common discrepancies in genome annotation, one SORF resulted from a rare start codon (AUA) as the initiation site for translation of this protein. Also, a methylation on a 30S ribosomal protein (MA1259) was localized to Pro59-Val69, contrasting sharply from its homologue in Escherichia coli (rp S12) known to harbor an unusual beta-thiomethylated aspartic acid residue.

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Figures

Figure 1

Figure 1

Mercury ion binding protein exhibiting a C-X-X-C motif was detected eluting at ~25 min (a). LC-MS/MS identified the protein (b) with an e-value of 4 × 10−11 (c). A disulfide bond was observed producing a −2 Da mass discrepancy. Thioredoxin has a C-X-X-C motif likely for facilitating reduction. Furthermore, this protein has a mispredicted start site (d), where the first four amino acids have been incorrectly annotated. The most abundant fragment ions have been labeled in (b).

Figure 2

Figure 2

Several proteins were detected whose genes are present on the chromosome in a putative operon in an LC-MS run (a). Using CID to produce b- and y-ions, each protein was identified with a mispredicted start site, (b)–(e).

Figure 3

Figure 3

A protein was identified despite not being annotated in the M. acetivorans database. In (a), 25 CID fragmentation scans of the LC-MS target were acquired via automated nanospray. A biomarker search of a database created from simple six-frame translations revealed a single hit, (b). DNA sequencing revealed that a Met (AUA) was the start codon for translation initiation. Four more unannotated proteins were identified using a biomarker search of a six-frame translated database, (c)–(f).

Figure 4

Figure 4

Intact protein MA1259 ribosomal protein S12P exhibits a +13.98 Da mass shift. Presumably, the uncalibrated data from the Q-FT mass spectrometer, which lacks automatic gain control, produced a −0.04 Da (−7.92 ppm) discrepancy from the theoretical +14.02 Da methylation. The protein was fragmented with ECD to localize the modification to a stretch of 30 amino acids (a). Offline analysis of a Glu-C digest detected a peptide with two missed cleavages incorporating the +14.02 Da modification (b). Fragmentation data further narrowed down possible sites of modification to 28 amino acids.

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