Application Note: 498 Utilizing a Hybrid Mass Spectrometer to Enable Fundamental Protein Characterization: Intact Mass Analysis and Top-Down Fragmentation with the LTQ Orbitrap MS Tonya Pekar Second, Vlad Zabrouskov, Thermo Fisher Scientific, San Jose, CA, USA Alexander Makarov, Thermo Fisher Scientific, Bremen, Germany Introduction Experimental Key Words A fundamental stage in protein characterization is to Protein standards, including bovine carbonic anhydrase, • LTQ Orbitrap Velos determine and verify the intact state of the macromolecule. yeast enolase, bovine transferrin and human monoclonal This is often accomplished through the use of mass IgG, were purchased from Sigma-Aldrich. For direct • LTQ Orbitrap XL spectrometry (MS) to first detect and measure the molecular infusion, proteins in solution were purified by either a • Applied mass. Beyond confirmation of intact mass, the next objective Thermo Scientific Vivaspin centrifugal spin column or a Fragmentation is the verification of its primary structure, the amino acid size-exclusion column (GE Healthcare), employing at least Techniques sequence of the protein. Traditionally, a map of the two rounds of buffer exchange into 10 mM ammonium macromolecule is reconstructed from matching masses of acetate. Protein solutions were at a concentration of least • Electron Transfer peptide fragments produced through external enzymatic 1 mg/mL prior to clean-up. Samples were diluted into Dissociation ETD digestion of the protein to masses calculated from an in 50:50:0.1 acetonitrile:water:formic acid prior to infusion silico • Top-Down digest of the target protein sequence. A more direct into the mass spectrometer. Instrument parameters were approach involves top-down MS/MS of the intact protein altered during infusion of protein solutions to optimize the Proteomics molecular ion. signal-to-noise ratio and the accuracy of measurement. For To accommodate progressive stages of characterization, LC/MS analysis, a polystyrene di-vinyl benzene (PSDVB) the choice of instrument for MS-based protocols should protein microtrap (Michrom Bioresources) was utilized to consider the diversity of required analyses, including further desalt proteins on-line. interrogation of protein sequence modifications, which can Experiments were performed on LTQ Orbitrap XL ™ be very labile (glycosylation, phosphorylation). A hybrid and LTQ Orbitrap Velos ™ hybrid mass spectrometers. linear ion trap-Orbitrap mass spectrometer offers extended Top-down experiments were conducted using direct infusion versatility for this application, featuring both high-performance of a 5 pmol/uL solution of enolase. All data were acquired detection (high resolution, mass accuracy, and dynamic with external mass calibration. The Xtract feature within range) and distinctive flexibility in operation. 1-6 It offers Thermo Scientific Xcalibur software was used to deconvolute several choices of fragmentation, to permit sophisticated isotopically resolved spectra to produce a monoisotopic experiments such as post-translational modification (PTM) mass peak list for high-resolution MS and MS/MS data. analysis with preservation of labile modifications (electron Thermo Scientific ProMass Deconvolution software was transfer dissociation, ETD) ,3-35 de novo sequencing (high used to deconvolute intact protein spectra that contained mass accuracy and combined activation types) ,36-39 and unresolved isotopic clusters in production of an average disulfide mapping to augment superior, sensitive MS/MS zero charge mass of the intact protein. Thermo Scientific detection for peptide identification. 40-48 ProSightPC 2.0 software was used to map top-down frag - This note describes protein characterization using the mentation spectral data to associated sequences utilizing Thermo Scientific LTQ Orbitrap XL or LTQ Orbitrap the single-protein mode search. Deconvoluted spectra Velos hybrid mass spectrometer, demonstrating intact protein produced from the Xtract algorithm within Xcalibur soft - and top-down fragmentation analysis (Figure 1). 48-58 ware were imported into ProSightPC software. To account for errors in the determination of the monoisotopic mass, a parameter tolerance of 1.01 Da was used to match frag - ments. However, only those fragments which correspond - ed to a mass error of 1.0076 Da ± 5 ppm were considered correct. Figure 1: Instrumental schematic of the LTQ Orbitrap XL ETD mass spectrometer, indicating the available fragmentation mechanisms Results and Discussion Mass Spectrometry – Intact Analysis Sample Preparation The mass spectrum of large macromolecules is a composite of a distribution of naturally-occurring isotopes. As molecules Sample preparation prior to electrospray ionization (ESI) increase in mass, the contribution of higher mass isotopes is critical for the generation of usable mass spectra, (13 C) increases proportionally. For large proteins, the natural especially for intact protein analysis. A high concentration distribution of these isotopes is nearly Gaussian, with the of salt or other adducts can suppress ionization and there - most abundant isotopes adjacent to the average mass of the fore sensitivity of analysis. It can also complicate spectra, protein. Electrospray ionization (ESI) produces a series of broadening the observed MS peak to reduce sensitivity multiply-charged ions detectable by the mass spectrometer through division of analyte signal among ions of heteroge - (Figure 2). Depending on the specification of the mass neous composition. spectrometer, the isotopes of each charge state may or may Sample purification can be accomplished through both not be resolved. The LTQ Orbitrap XL or LTQ Orbitrap on-line LC/MS chromatographic separation from salt or Velos hybrid mass spectrometer is capable of resolving off-line clean-up followed by direct infusion. For protein power in excess of 100,000 (FWHM) at m/z 400. This mixtures, proteins should be chromatographically resolved allows isotopic resolution of proteins close to 50,000 Da. as much as possible to maximize MS detection of individual For best analysis of large macromolecules, it was compounds. It is optional to use formic acid as a modifier determined that the initial portion of the FT transient for reverse-phase (RP) separations to avoid ionization produced during image current detection (containing first suppression induced by trifluoroacetic acid (TFA), though beat) contained the largest signal for transformation of TFA can also improve the concentration of protein elution large macromolecules with Orbitrap analyzer detection. This during chromatographic separation which can improve necessitates a reduction in the delay between excitation signal to noise for LC/MS separations. of the ion population (injection of ion cloud into the Orbitrap mass analyzer) and detection of the transient. To accomplish this on LTQ Orbitrap, LTQ Orbitrap XL, and LTQ Orbitrap Discovery mass spectrometers, a toggle for analysis of high-molecular- weight molecules is accessible from the instrument control window (Tune page) in instru - ment control software version 2.5.5 SP1 or later. The toggle is located under the diagnostics file menu under Tools → Toggles → FT detection delay, and should be set to High for large macromolecules. No changes are necessary for the LTQ Orbitrap Velos instru - ment. If isotopic resolution of a protein is desired and achiev - able, a higher resolution setting should be used for detection – 60,000 or 100,000 resolving power. It is recommended that the pressure in the Orbitrap analyzer be less than 2 ¥10 -10 Torr. For larger proteins for which isotopic resolution is possible, it is recommended that sufficient spectral averag - ing is conducted to produce the best isotopic envelope, espe - cially for broadband acquisi - tion. Figure 2: Mass spectrum of bovine carbonic anhydrase with one acetylation modification (29006.682 Da), depicting isotopic resolution of the z = 34 charge state. Data was acquired at 100,000 resolving power with Orbitrap analyz - er detection. Accurate mass of the most abundant isotope is 1.73 ppm. HCD gas was turned off for acquisition on the LTQ Orbitrap XL instrument. Source voltage of 10 V was applied. Figure 3: Mass spectrum from LTQ Orbitrap XL mass spectrometer yeast enolase (monoisotopic mass of 46642.214 Da) with broadband acquisition, depicting isotopic resolution. Inset depicts the z = 45 charge state with an accurate mass of 0.12 ppm for the most abundant isotope. Data was acquired at 100,000 resolving power with broadband Orbitrap analyzer detection ( m/z 900-1200 shown), averaging ~600 scans. HCD gas was turned off for acquisition with the LTQ Orbitrap XL instrument. A tube lens of 110 V (LTQ Orbitrap XL instrument) or S-lens of 50% (LTQ Orbitrap Velos instrument) was used in combination with 10 V of source voltage. Alternatively, a subset of charge states can be isolated in the ion trap and subjected to Orbitrap analyzer detection to increase ion statistics for production of a better quality isotopic envelope, potentially requiring less spectral averaging. Figure 3 depicts the mass spectrum of the z = 45 charge state of yeast enolase with isotopic resolution. For proteins for which isotopic resolution cannot be achieved (i.e. proteins >50 kDa), it is recommended to run at the 7500 or 15,000 resolution setting of the Orbitrap analyzer due to the interference effects as described in Makarov et al. 53 This results in a better signal-to-noise ratio and, because of the higher scan rate, allows for more scans averaged across a chromatographic