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Home , Hybrid mass spectrometer, Mass spectrum, Monoisotopic mass, Orbitrap, Tandem mass spectrometry

Application Note: 498 Utilizing a Hybrid to Enable Fundamental Characterization: Intact Mass Analysis and Top-Down Fragmentation with the LTQ 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, 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 • Transfer 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 molecular . 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 . linear -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 , 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 , 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 – Intact Analysis Sample Preparation The mass of large macromolecules is a composite of a distribution of naturally-occurring . As Sample preparation prior to electrospray (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. (ESI) produces a series of broadening the observed MS peak to reduce sensitivity multiply-charged 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: 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 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 peak (Figure 4). The accuracy of mass measurement for unresolved isotopic clusters is not dependent on the resolution setting. It was also found that S/N is improved when Figure 4: Mass spectrum of enolase acquired at 7500 resolving power. The spectrum is an average of the supply of external gas to the HCD collision cell is ~100 scans across a chromatographic peak of 2 µg of enolase injected on a protein microtrap. Mass accuracy reduced. The collision gas can be turned off in the of the deconvoluted average mass (46670.6879 experimental) is 2.4 ppm. HCD gas was turned off for diagnostics file menu within the instrument control 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. window (tune software), under Tools → Toggles → FT increased upwards to promote a cleaner signal. For large HCD collision gas. The toggle should be set to “off”. This macromolecules such as intact (~150 kDa), a toggle is available for the LTQ Orbitrap XL mass spec - source voltage of >35 V is recommended. It should be trometer. Due to a different layout of gas delivery, the noted that the fragility of protein macromolecules and LTQ Orbitrap Velos mass spectrometer offers a lower covalent modifications can vary. Too much energy deposi - Orbitrap analytical pressure than previous systems, and tion can also result in loss of fragile modifications or in the HCD gas does not need to be turned off. fragmentation of the parent . As discussed previously, mass spectral protein signal is Some adjustment of parameters, such as reduction of often complicated by the association of non-covalent source voltage, temperature, or tube lens/S-lens voltage, may adducts such as salt, acid and solvent adducts. It is best be required to minimize fragmentation. Alternatively, if a practice to achieve efficient desolvation of the protein protein spectrum is pure, a high source voltage can be used upon electrospray ionization in addition to separation to fragment the intact molecular ion (top-down analysis). from salt. The sheath and auxiliary gas in the ESI source in This approach has the advantage of subjecting all charge conjunction with increased temperatures (capillary tem - states to fragmentation, which improves signal for the perature, heated electrospray probe) aid in desolvation. MS/MS spectrum, as the sum of signal from all contributing These values should be set appropriate to the LC flow charge states is utilized. This also permits access to alternative rate, and can be tuned manually with infusion of the ana - fragmentation pathways from different charge states. A lyte into the desired LC flow rate. During infusion of pro - source voltage of up to 100 V can be applied to induce tein solutions for optimization of acquisition parameters, fragmentation of all ions and all charge states. it is recommended that the number of microscans be The voltage of the tube lens in LTQ Orbitrap XL or increased to 5 or more for the full scan. Tuning of the Discovery instruments, and the S-lens in LTQ Orbitrap source parameters should be done using the ion trap Velos instruments affects ion transmission to the mass detector. analyzer within a desired mass range. For proteins which The application of source voltage (SID) can also be ionize at higher m/z , a higher lens voltage is required. used to aid in desolvation or removal of undesired These values can be adjusted manually. For large macro - adducts. The amount of source voltage to apply is dependent molecules such as antibodies, the required voltage can be on the size and stability of the protein and also on the in excess of 200 V (tube lens) or 80% (S lens). Other ion strength of attraction between protein ions and adducts. optic voltages were found to be much less influential. A setting of 10-20 V is a good starting point, and can be Deviation from default values influenced signal less than 30% for the proteins analyzed in these experiments. It is recommended that they be left at default or as set during tuning with a small protein (ex: myoglobin). For molecules such as intact antibodies analyzed in the high-mass range (up to m/z 4000), an additional calibration step is required in order to achieve proper mass accuracy with Orbitrap analyzer detection. This adjusts the ion population to the desired target when ions are ejected from the to the FTMS in high-mass mode, and does so by adjusting the calculated injection time. This procedure is also located in diagnostics, under Toggles → System evaluation → FT high mass range target compensation. Figure 5 depicts the mass spectrum for an immunoglobulin (IgG), with associated acquisition parameters listed. Again, a higher tube lens or S-lens voltage also promotes transmission of higher m/z ions for analysis in the high-mass range.

Figure 5: Mass spectrum of intact immunoglobulin (IgG, 147,251 Da) detected at 7500 RP by LC/MS. Mass accuracy of the average mass was 6 ppm. A tube lens of 230 V (LTQ Orbitrap XL instrument) or S-lens 90% (LTQ Orbitrap Velos instrument) was used. Source voltage of 40 V was used to remove adducts. HCD gas was turned off for the LTQ Orbitrap XL mass spectrometer. Mass Spectrometry – Top-Down Analysis Collision-induced dissociation (CID) induces fragmen - Beyond routine intact mass verification lies the need to tation through resonance excitation in the linear ion trap determine or confirm the identity of a protein’s primary followed by detection of fragments in the Orbitrap mass amino acid sequence. As described, the hybrid linear ion analyzer. Resonance excitation prevents excitation of trap – Orbitrap system allows multiple activation strategies, daughter ions that would induce secondary or tertiary which often provide complimentary information. The fragmentation pathways, and has the advantage of limiting hybrid system can also resolve complex fragmentation the production of internal fragments. Figure 6 shows the spectra from high-molecular-weight precursors, which CID MS/MS spectrum for yeast enolase, isolated m/z 100 contain many fragments of large mass. window around m/z 1010. Whole protein fragmentation has the advantage of For Orbitrap mass analyzer detection, the HCD collision circumventing the limitations of traditional peptide mapping cell of the LTQ Orbitrap Velos mass spectrometer provides (such as in vitro -induced modifications, <100% sequence a significant improvement in the efficiency of ion extraction coverage and loss of information of the intact state). after fragmentation, especially for molecular ions of higher Alternate charge states of the same precursor can follow charge. Here, lower normalized collision energy resulted different fragmentation pathways to produce alternate in more-optimal fragmentation (larger fragments) in the fragments (different b/y or c/z ion pairs). For fragmenta - HCD collision cell. Excessively high collision energy resulted tion of a pure protein, it is possible to isolate a range of in over-fragmentation. For HCD, the voltage offset that charge states and subject all to fragmentation by CID, should be applied versus the collision cell is dependent on HCD or ETD. For example, an isolation width of m/z 100 the charge state of the precursor (collision energy is a nor - was employed here to isolate multiple charge states. malized value in consideration of precursor charge, when Because the initial signal is divided among multiple the charge state is specified or determined by the system). fragments, multiple charge states and multiple isotopes For large proteins for which the instrument cannot deter - within those charge states, spectral averaging is required mine the charge (not resolved), the assumed charge state to achieve best sequence coverage with top-down fragmen - can be chosen and input into the tune page for acquisi - tation. tion. A collision energy of 13 was used for fragmentation in the HCD collision cell of an LTQ Orbitrap Velos mass spectrometer, isolating a m/z 100 window around m/z 850 and a specified charge state of 50.

Figure 6: CID MS/MS spectrum for yeast enolase from an LTQ Orbitrap Velos mass spectrometer acquired at 100,000 resolving power, isolating a m/z 100 window around m/z 1010 with a normalized collision energy of 30. The inset shows resolution of large sequence fragments (y221 and y222) with masses greater than 20 kDa. Mass accuracy of the monoisotopic peaks for the labeled fragments, upon deconvolution with Xtract software, was less than 1 ppm. Approximately 2000 scans were averaged. Electron transfer dissociation (ETD) is a kinetic reaction The combination of information from alternative with the rate dependent on the charge of the precursor – fragmentation strategies produced the most comprehensive the higher the charge state, the faster the reaction occurs. coverage of the protein sequence. The sequence map shown Shorter activation times should be used for highly-charged in Figure 8 illustrates the advantage of combining infor - proteins to produce larger fragments. Activation time can mation garnered from multiple activation types to define also be manipulated to influence coverage in alternate the amino acid sequence, offering amino acid resolution regions of the protein sequence. For example, a longer for large parts of the N and C termini of the sequence. activation time can influence the production of lower-charge Complementary fragmentation mechanisms also provide fragments that correspond to the N- or C-terminus of a increased evidence to confirm bond linkages, with the protein sequence. 48 Spectra were collected for alternative presence of b/y or c/z-type ions for the same breakage activation times – including 4 ms, 7 ms, 15 ms, 50 ms, point, or coverage in alternative regions of the sequence. and 100 ms. The shorter activation times resulted in richer CID produced larger fragments representing cleavage in fragmentation spectra, while longer activation times pro - the middle of the sequence. Preservation of the intact state duced fragments corespondent to the termini of the pro - of the molecule through whole-molecule fragmentation tein sequence. Figure 7 displays ETD MS/MS fragmenta - avoids loss of information, which is a common consequence tion spectra produced using a 4 msec (A) and 100 msec of protein digestion used in traditional peptide mapping activation time (B). characterization.

Figure 7: ETD MS/MS spectrum of enolase acquired at 60,000 resolving power, isolating a m/z 100 window around m/z 790, with an activation time of 4 msec. Approximately 1000 scans were averaged (A). ETD MS/MS spectrum of enolase acquired at 60,000 resolving power, isolating a m/z 100 window around m/z 790, with an activation time of 100 msec. Approximately, 600 scans were averaged (B). Some example sequence ions which could be labeled in the space of the figure are shown, with corresponding mass accuracies. A detailed map of coverage can be seen in Figure 8. References 1. Makarov, A.; Denisov, E.; Kholomeev, A.; Balschun, W.; Lange, O.; Strupat, K.; Horning, S. Performance Evaluation of a Hybrid Linear Ion Trap/Orbitrap Mass Spectrometer. Anal. Chem. 2006, 78 (7), 2113-2120. 2. Makarov, A.; Denisov, E.; Lange, O.; Horning, S. Dynamic Range of Mass Accuracy in LTQ Orbitrap Hybrid Mass Spectrometer. J. Am. Soc. Mass Spectrom. 2006, 17 (7), 977-982. 3. 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