Top-down analysis of small plasma proteins using an LTQ-Orbitrap. Potential for mass spectrometry-based clinical assays for transthyretin and hemoglobin

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Abstract

Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where point mutation characterization is diagnostically critical. We have developed a top-down analytical platform for variant and/or modified protein sequencing and are examining the feasibility of using this platform for the analysis of hemoglobin/TTR patient samples and evaluating the potential clinical applications. The platform is based on a commercial high resolution hybrid orbitrap mass spectrometer (LTQ-Orbitrap™) with automated sample introduction; automated data analysis is performed by our own software algorithm (BUPID top-down).

The analytical strategy consists of iterative data capture, first recording a mass profile of the protein(s). The presence of a variant is revealed by a mass shift consistent with the amino acid substitution. Nozzle-skimmer dissociation (NSD) of the protein(s) yields a wide variety of sequence-defining fragment ions. The fragment ion containing the amino acid substitution or modification can be identified by searching 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 supplemented 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.

Research highlights

▶ Top-down sequencing of proteins for detection of variants in plasma proteins. ▶ Post-translational modifications also defined and located. ▶ Nozzle-skimmer dissociation followed by CID. ▶ Approach should have potential for clinical use.

Introduction

Current approaches to protein structure determination by mass spectrometry center on so-called bottom-up and top-down approaches. Bottom-up methods [1], [2] utilize proteolytic digestions to break down the protein into constituent peptides that can be readily analyzed by LC–MS/MS whereby separation and dissociation of the peptides provides structural information for many peptides in a single experiment. The disadvantages associated with the bottom-up approach are: labor intensive and time consuming sample preparation, together with less than 100% sequence coverage, as well as the potential for introduction of artifacts during digestion. Furthermore, molecular weight information lost through proteolysis hinders the search for post-translational modifications and information on the relationship(s) among multiple post-translational modifications (PTMs) to a single protein is usually sacrificed.

For the top-down approach, proteins are directly introduced into the mass spectrometer and individual components can be mass-selected and dissociated in the instrument, yielding product ions containing structural information. Top-down [3], [4] is also more efficient in its use of mass spectrometer system time because separations can be performed off-line if necessary. The methodology of top-down analysis circumvents the drawbacks listed above by analyzing intact protein molecular weights and fragments ions directly within the mass spectrometer. Although bottom-up remains the workhorse of proteomics, the characterization of point mutations by LC–MS/MS can be made difficult by the higher false positive rate that is observed when sequence variations are allowed in the database search.

Peptide mapping was demonstrated to be useful for hemoglobin variant determination by fast atom bombardment mass spectrometry [5]. The use of targeted analysis for common hemoglobin 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 primary structural information from intact proteins through MS/MS on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer [7], [8] originated soon after the introduction electrospray ionization mass spectrometry. McLuckey et al. [9] have adapted ion traps for the purpose of top-down protein sequencing. Pseudo MS3 analyses using quadrupole orthogonal time-of-flight (Q-o-TOF) instruments were reported by Thevis et al. [10] for top-down determinations on transferrins. A triple quadrupole instrument was used by Witkowska et al. to characterize a genetic mutation that gave rise to a variant human α-globin [11]. Whilst a variety of instrumental configurations have thus been used for the top-down analysis of proteins, the high resolution, mass accuracy and MSn capacity of FTICR MS [12], [13], as well as the availability of multiple activation techniques, including infrared multiphoton dissociation (IRMPD) [14], electron capture dissociation (ECD) [15], electron transfer dissociation (ETD) [16], and sustained off-resonance collisionally activated dissociation (SORI-CAD) [17], continue to favor these instruments for top-down analysis.

The recent introduction of the LTQ-Orbitrap mass spectrometer [18] offers many of the high resolution, mass accuracy and MSn advantages of FTICR-MS at a more affordable price. Macek et al. [19] demonstrated the ability of the LTQ-Orbitrap to generate top-down data using protein standards ranging from 10 to 25 kDa. However, their tandem analysis was performed exclusively within the linear ion trap portion of the system. The multistage MS/MS capability of the system was not investigated. The LTQ-Orbitrap has similarly been used in a top-down mode to characterize recombinant monoclonal antibodies [20], [21].

Whilst these examples highlight the potential of the LTQ-Orbitrap for the top-down analysis of proteins, a comprehensive approach that fully exploits the high performance capabilities of this hybrid mass spectrometer has not been presented. This approach could be particularly useful in the case of small and easily isolated plasma proteins of clinical relevance such as transthyretin and hemoglobins, where characterization of amino acid substitutions that can define genetic point mutations integral to the pathogenesis of the diseases is necessary for precise diagnosis of hereditary transthyretin amyloidosis [22] and hemoglobinopathies [23], respectively. The use of mass spectrometry to characterize variants of these proteins has traditionally relied on bottom-up approaches [5], [24], [25], [26]. A protocol that combines intact protein molecular weight profiling by FT-ICR MS with DNA sequencing has been implemented for the detection and characterization of TTR variants [27], [28] in a clinical setting. The relatively small size, ease of isolation in abundant quantities, and absence of structural features refractory to collisional activation (e.g., no intramolecular disulfide bonds) make TTR and hemoglobins ideal candidates for top-down analysis. However, to our knowledge, transthyretin-related proteins have only been analyzed using top-down in our published study of TTR fragments ranging from 7 to 10 kDa that were extracted from amyloid fibrils and sequenced with a Q-o-TOF MS [29].

Top-down MS/MS has been used for hemoglobins; indeed, they were among the first proteins to be analyzed in this manner [7], [8], [30]. Nevertheless, top-down MS/MS has only been applied to hemoglobin variant characterization in very few instances. The potential of top-down MS/MS for the characterization of hemoglobinopathies was recognized in 1996 but the complexity of spectra, unresolved charge state ambiguities and the lack of suitable software tools for spectra interpretation were cited as obstacles for the routine application of the method. In their review on the use of mass spectrometry in the hemoglobinopathy field published at this time, these researchers pointed out the desirability of developing top-down methods for the sequencing of intact hemoglobin chains [31].

The preliminary study whose results are reported herein aimed to explore the applicability of a simple top-down method to TTR and hemoglobin variant characterization. By combining automated sample introduction, LTQ-Orbitrap and customized software algorithms written in-house, an analytical platform was assembled and tested. The results presented in this preliminary study highlight the feasibility of this approach for characterizing variants of transthyretin and hemoglobins. The potential for this method to be applied in a clinical setting seems promising. Given that the LTQ-Orbitrap system is becoming quite widely available, it is important to raise the awareness of the clinical community to the potential of these instruments for straightforward top-down analyses.

Section snippets

Samples

Transthyretin was obtained by immunoprecipitation from human serum, using the method outlined by Lim et al. [32] with the difference that a smaller volume of serum was used (50 μL vs. 200 μL). Hemoglobin was obtained from whole blood by diluting 1 μL of whole blood in 500 μL of ESI buffer. Use of de-identified patient samples was approved by BUSM. All samples were analyzed in ESI buffer composed of 50% water, 50% acetonitrile containing 0.2% formic acid. Sample concentration was approximately 1 

General comments

The basic analytical strategy consists in gathering a maximum of structural information by obtaining molecular weight data on the intact protein(s) and then dissociating the molecular species to generate complementary ion pairs that have sufficient abundances and appropriate masses for subsequent further stages of tandem MS. In theory, given that the LTQ-Orbitrap offers three potential sites for ion activation (the nozzle-skimmer region, the linear ion trap (LTQ) and C-trap), multiple

Conclusions

The combination of automated sample introduction, the high resolution and high mass accuracy of the hybrid LTQ-Orbitrap and customized software algorithms written in-house (BUPID top-down) provides an integrated analytical platform amenable to the analysis of TTR and hemoglobin variants and their post-translational modifications. The method easily lend itself to automation for use in clinical laboratories. Two limitations should be noted: (1) the method achieves high sequence coverage but does

Acknowledgements

This research is supported by NIH-NCRR grants P41 RR010888 and S10 RR020946 and by NIH-NHLBI contract N01 HV028178. Transthyretin samples were supplied by the BUSM Amyloid Treatment and Research Program. Samples containing variant hemoglobins were provided by the BUSM Sickle Cell Center.

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