Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without harvesting the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier transform ion cyclotron resonance (FTICR) to analyze macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation.
SummaryGenes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 ∞ C allowed formation of an a 7 b 7 b 7 a 7 , 750 kDa cylindrical stack of four rings in which all 14 b -subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete , Rhodococcus erythropolis , Mtb's b -chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg 2 + or Ca 2 + . Electron microscopy revealed what appeared to be obstructed a -rings in the Mtb 750 kDa particle. Deletion of the Nterminal octapeptide from Mtb's a -chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes , the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.
The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the a -subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapetides of the a -subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N -(4-morpholine)carbonyl-b -(1-naphthyl)-Lalanine-L -leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtbspecific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) delivers high resolving power, mass measurement accuracy, and the capabilities for unambiguously sequencing by a top-down MS approach. Here, we report isotopic resolution of a 158 kDa protein complex - tetrameric aldolase with an average absolute deviation of 0.36 ppm and an average resolving power of ~520,000 at m/z 6033 for the 26+ charge state in magnitude mode. Phase correction further improves the resolving power and average absolute deviation by 1.3 fold. Furthermore, native top-down electron capture dissociation (ECD) enables the sequencing of 149 C-terminal amino acid (AA) residues out of 463 total AAs. Combining the data from top-down MS of native and denatured aldolase complexes, a total of 58% of the backbone cleavages efficiency is achieved. The observation of complementary product ion pairs confirms the correctness of the sequence and also the accuracy of the mass fitting of the isotopic distribution of the aldolase tetramer. Top-down MS of the native protein provides complementary sequence information to top-down ECD and CAD MS of the denatured protein. Moreover, native top-down ECD of aldolase tetramer reveals that ECD fragmentation is not limited only to the flexible regions of protein complexes and that regions located on the surface topology are prone to ECD cleavage.
“Native” mass spectrometry (MS) has been proven increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD MS hADH dimer shows that each subunit (E and S chain) binds not only to two zinc atoms, but also the NAD+/NADH ligand, with a higher NAD+/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.
The yeast mitochondrial protein Suv3p is a putative NTP-dependent RNA helicase. Here we report that in cells lacking Suv3p, there is an approximately 50-fold increase in the excised form of the group I intron omega of the mitochondrial 31S rRNA gene. Surprisingly, little mature 21S rRNA accumulates in those cells; instead, unligated 21S rRNA exons appear. Intron overaccumulation could lead to spliced exon reopening via a reaction known to be catalyzed by group I introns in vitro. We also show that Suv3p is a functional component of a novel mitochondrial NTP-dependent 3'-to-5' exoribonuclease activity that can degrade group I intron RNAs. These findings account for group I intron overaccumulation in cells lacking Suv3p and define a novel function for putative RNA helicases in direct RNA degradation.
A high resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is used for characterizing the fragmentation of chlorophyll-a. Three tandem mass spectrometry (MS/MS) techniques, including electron-induced dissociation (EID), collisionally activated dissociation (CAD), and infrared mutiphoton dissociation (IRMPD) are applied to the singly protonated chlorophyll-a. Some previously unpublished fragments are identified unambiguously by utilizing high resolution and accurate mass value provided by the FTICR mass spectrometer. According to this research, the two long aliphatic side chains are shown to be the most labile parts, and favorable cleavage sites are proposed. Even though similar fragmentation patterns are generated by all three methods, there are much more abundant peaks in EID and IRMPD spectra. The similarities and differences are discussed in detail. Comparatively, cleavage leading to odd electron species and H(•) loss both seem more common in EID experiments. Extensive loss of small side groups (e.g., methyl and ethyl) next to the macrocyclic ring was observed. Coupling the high performance FTICR mass spectrometer with contemporary MS/MS techniques, especially IRMPD and EID, proved to be very promising for the structural characterization of chlorophyll, which is also suitable for the rapid and accurate structural investigation of other singly charged porphyrinic compounds.
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