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Top-Down Analysis of Small Plasma Proteins Using an LTQ-Orbitrap

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Top-Down Analysis of Small Plasma Proteins Using an LTQ-Orbitrap International Journal of Mass Spectrometry 300 (2011) 130–142 Contents lists available at ScienceDirect International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms Top-down analysis of small plasma proteins using an LTQ-Orbitrap. Potential for mass spectrometry-based clinical assays for transthyretin and hemoglobin Roger Théberge, Giuseppe Infusini, Weiwei Tong, Mark E. McComb, Catherine E. Costello ∗ Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA 02118, USA article info abstract Article history: Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where Received 20 April 2010 point mutation characterization is diagnostically critical. We have developed a top-down analytical Received in revised form 10 August 2010 platform for variant and/or modified protein sequencing and are examining the feasibility of using Accepted 17 August 2010 this platform for the analysis of hemoglobin/TTR patient samples and evaluating the potential clinical Available online 24 August 2010 applications. The platform is based on a commercial high resolution hybrid orbitrap mass spectrometer (LTQ-OrbitrapTM) with automated sample introduction; automated data analysis is performed by our Keywords: own software algorithm (BUPID top-down). Top-down Protein The analytical strategy consists of iterative data capture, first recording a mass profile of the protein(s). Variant The presence of a variant is revealed by a mass shift consistent with the amino acid substitution. Nozzle- Transthyretin skimmer dissociation (NSD) of the protein(s) yields a wide variety of sequence-defining fragment ions. Hemoglobin The fragment ion containing the amino acid substitution or modification can be identified by searching Post-translational modification for a peak exhibiting the mass shift observed in the protein mass profile. This fragment ion can then be selected for MS/MS analysis in the ion trap to yield sequence information permitting the identification of the variant. Substantial sequence coverage has been obtained in this manner. This strategy allows for a stepwise MS/MS analysis of the protein structure. The sequence information obtained can be supple- mented with whole protein NSD fragmentation and MS/MS analysis of specific protein charge states. The analyses of variant forms of TTR and hemoglobin are presented to illustrate the potential of the method. © 2010 Elsevier B.V. All rights reserved. 1. Introduction For the top-down approach, proteins are directly introduced into the mass spectrometer and individual components can be Current approaches to protein structure determination by mass-selected and dissociated in the instrument, yielding product mass spectrometry center on so-called bottom-up and top-down ions containing structural information. Top-down [3,4] is also more approaches. Bottom-up methods [1,2] utilize proteolytic digestions efficient in its use of mass spectrometer system time because sepa- to break down the protein into constituent peptides that can be rations can be performed off-line if necessary. The methodology of readily analyzed by LC–MS/MS whereby separation and dissoci- top-down analysis circumvents the drawbacks listed above by ana- ation of the peptides provides structural information for many lyzing intact protein molecular weights and fragments ions directly peptides in a single experiment. The disadvantages associated with within the mass spectrometer. Although bottom-up remains the the bottom-up approach are: labor intensive and time consum- workhorse of proteomics, the characterization of point mutations ing sample preparation, together with less than 100% sequence by LC–MS/MS can be made difficult by the higher false positive coverage, as well as the potential for introduction of artifacts rate that is observed when sequence variations are allowed in the during digestion. Furthermore, molecular weight information lost database search. through proteolysis hinders the search for post-translational mod- Peptide mapping was demonstrated to be useful for hemoglobin ifications and information on the relationship(s) among multiple variant determination by fast atom bombardment mass spectrom- post-translational modifications (PTMs) to a single protein is usu- etry [5]. The use of targeted analysis for common hemoglobin ally sacrificed. variants through multiple reaction monitoring (MRM) acquisition mode MS of whole blood tryptic digests was proposed recently as a population screening methodology [6]. The idea of generating pri- ∗ mary structural information from intact proteins through MS/MS Corresponding author at: Center for Biomedical Mass Spectrometry, Department on a Fourier transform ion cyclotron resonance (FT-ICR) mass spec- of Biochemistry, Boston University School of Medicine, 670 Albany Street, Room 511, Boston, MA 02118-2646, USA. Tel.: +1 61 7 638 6490; fax: +1 61 7 638 6491. trometer [7,8] originated soon after the introduction electrospray E-mail address: [email protected] (C.E. Costello). ionization mass spectrometry. McLuckey et al. [9] have adapted 1387-3806/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijms.2010.08.012 R. Théberge et al. / International Journal of Mass Spectrometry 300 (2011) 130–142 131 ion traps for the purpose of top-down protein sequencing. Pseudo and tested. The results presented in this preliminary study high- MS3 analyses using quadrupole orthogonal time-of-flight (Q-o- light the feasibility of this approach for characterizing variants of TOF) instruments were reported by Thevis et al. [10] for top-down transthyretin and hemoglobins. The potential for this method to be determinations on transferrins. A triple quadrupole instrument was applied in a clinical setting seems promising. Given that the LTQ- used by Witkowska et al. to characterize a genetic mutation that Orbitrap system is becoming quite widely available, it is important gave rise to a variant human ␣-globin [11]. Whilst a variety of to raise the awareness of the clinical community to the potential of instrumental configurations have thus been used for the top-down these instruments for straightforward top-down analyses. analysis of proteins, the high resolution, mass accuracy and MSn capacity of FTICR MS [12,13], as well as the availability of multiple 2. Experimental activation techniques, including infrared multiphoton dissociation (IRMPD) [14], electron capture dissociation (ECD) [15], electron 2.1. Samples transfer dissociation (ETD) [16], and sustained off-resonance col- lisionally activated dissociation (SORI-CAD) [17], continue to favor Transthyretin was obtained by immunoprecipitation from these instruments for top-down analysis. human serum, using the method outlined by Lim et al. [32] with The recent introduction of the LTQ-Orbitrap mass spectrome- the difference that a smaller volume of serum was used (50 ␮L vs. n ter [18] offers many of the high resolution, mass accuracy and MS 200 ␮L). Hemoglobin was obtained from whole blood by diluting advantages of FTICR-MS at a more affordable price. Macek et al. [19] 1 ␮L of whole blood in 500 ␮L of ESI buffer. Use of de-identified demonstrated the ability of the LTQ-Orbitrap to generate top-down patient samples was approved by BUSM. All samples were analyzed data using protein standards ranging from 10 to 25 kDa. However, in ESI buffer composed of 50% water, 50% acetonitrile contain- their tandem analysis was performed exclusively within the linear ing 0.2% formic acid. Sample concentration was approximately ion trap portion of the system. The multistage MS/MS capability 1 pmol/␮L. of the system was not investigated. The LTQ-Orbitrap has simi- larly been used in a top-down mode to characterize recombinant 2.2. Mass spectrometry monoclonal antibodies [20,21]. Whilst these examples highlight the potential of the LTQ- The samples were introduced into a “Classic” LTQ-Orbitrap mass Orbitrap for the top-down analysis of proteins, a comprehensive spectrometer (Thermo-Fisher Corp., Waltham, MA) using a TriVersa approach that fully exploits the high performance capabilities NanoMate system (Advion Biosciences, Ithaca, NY). A low-flow of this hybrid mass spectrometer has not been presented. This ESI chip was used as a static emitter to deliver the sample at approach could be particularly useful in the case of small and easily 20–40 nL/min. The LTQ-Orbitrap MS was operated in the nanospray isolated plasma proteins of clinical relevance such as transthyretin positive-ion mode. The top-down analyses were performed in a and hemoglobins, where characterization of amino acid substi- number of different activation modes in order to explore ion dis- tutions that can define genetic point mutations integral to the tribution and completeness of information on sequence and PTMs. pathogenesis of the diseases is necessary for precise diagnosis of Nozzle-skimmer dissociation was produced by varying the “source hereditary transthyretin amyloidosis [22] and hemoglobinopathies fragmentation” voltage between 60 and 100 V. For MS/MS analy- [23], respectively. The use of mass spectrometry to characterize sis, peaks with the m/z values of interest were selected within an variants of these proteins has traditionally relied on bottom-up isolation window width of m/z 4–8. Helium was employed as the approaches [5,24–26]. A protocol that combines intact protein collision gas for CID in the LTQ; analyses were performed at nor- molecular weight profiling by FT-ICR MS with DNA sequencing malized
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