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A Practical Introduction to Analysis of Intact Proteins on the TripleTOF™ 5600 System High Resolution and High Sensitivity for Analysis of Large Biomolecules

Doug Simmons AB SCIEX, Canada The analysis of intact proteins by accurate mass LC/MS is the method of choice for determining intact mass, protein heterogeneity, and post-translational modifications. Protein analysis demands MS hardware which offers high sensitivity, high resolution, and high mass accuracy across a wide mass range. The TripleTOF™ 5600 System meets all of these requirements and with a mass range up to m/z 40,000 allows for detection of proteins of all sizes. This technical note summarizes the practical aspects of intact protein analysis on the TripleTOF™ 5600 System, including mass calibration, method building/acquisition, and data analysis.

Key Steps for Obtaining High Quality Intact Protein Analysis using the TripleTOF™ 5600 System 1. Calibration

• LC AutoCalibration enables automated calibration for long term batch acquisitions

2. Acquisition

• Intact Protein Mode – automatically optimizes acquisition and signal processing algorithms specifically for intact protein analysis

3. Data Processing

• BioAnalyst™ Software provides multiple data processing tools for determining the intact protein mass from the LC/MS charge envelope

• PeakView™ Software provides computational tools for easy qualification of results

Figure 1. Analysis of Large Intact Proteins by LC/MS. An intact IgG protein can be easily analyzed by LC/MS on the TripleTOF™ 5600 System due to the extended mass range of the instrument (up to m/z 40,000). (Top) The charge state envelope is typically seen in the 2400 m/z region, due to high multiple charging of the ions. Analysis of this charge envelope provides a measure of the protein mass and information about PTMs (bottom).

1600 2000 2400 2800 3200 3600m/z

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Calibration Although mass calibration for intact protein analysis can be performed with the optional Calibrant Delivery System (CDS), it is more typically accomplished using the LC AutoCalibration feature. The LC AutoCalibration functions by performing a dedicated LC/MS calibration acquisition of a known reference solution periodically during a sample batch. The calibration acquisition employs a user-created method which encompasses the same mass range as the sample method, and may include a shortened gradient to reduce the time required for calibration. The calibration reference table includes the accurate m/z values of the reference ions as well as their corresponding retention times so the calibration can be performed automatically (Figure 2, top).

For optimum mass accuracy during intact protein analysis, it is recommended to calibrate using multiply-charged reference ions. The 6-Peptide Mixture, found in the LC/MS Peptide/Protein Mass Standards Kit1, contains peptides which yield several multiply charged peaks between m/z 419 – 1233 which is a sufficient range of reference ions for protein analysis (Figure 2, top). The calibration is linear in the m/z range of intact proteins so it is not of concern that the table of calibrant masses does not span the entire mass range of the acquisition.

For initial setup of LC AutoCalibration, an injection of the reference solution using the calibration acquisition method is performed to determine the retention times of the reference ions. Once the retention times are determined and entered into the reference table, LC AutoCalibration can be easily incorporated into sample analysis by selecting the appropriate reference table, calibration method, and reference solution vial on the Calibrate tab during batch submission.

Tip: Using XIC Manager in PeakView™ Software, the retention times of the six peptides can be easily determined (Figure 2, bottom). First, copy all the masses from the calibration reference table into XIC Manager. This allows the user to easily generate XICs on the LC/MS runs of the 6-Peptide Mixture. XIC Manager determines the retention times for each peptides, which can be subsequently copied back into the reference table.

In addition to providing automated mass calibration, LC AutoCalibration serves as a quality control acquisition which can be used to check for long-term consistency in chromatographic and MS performance over the course of a batch (Figure 3). The LC/MS Peak Statistics Utility (included in PeakView™ Software under the Help menu) provides an easy-to-use means of calculating several performance indicators (e.g. retention time, peak widths, mass accuracy, resolution, etc.) over multiple acquisitions.

Note: If instrument calibration using larger proteins is required in the workflow, this can be done through manual calibration. This will also enable a calibration to be performed with a Time Bins to Sum value closer to what will be used for the protein measurement when measuring very large proteins.

Figure 3: LC/MS Peak Statistics Utility. The LC/MS Peak Statistics Utility can be used to compare several key indicators of chromatographic and MS performance over multiple acquisitions. Here is shown the XIC height of calibrant ions from 6-Peptide Mixture over several LC AutoCalibration acquisitions.

Figure 2: LC AutoCalibration Using the 6-Peptide Mixture. (Top) TOF MS Calibration Reference table is used by LC AutoCalibration and contains the accurate m/z values and retention times for the 6-Peptide Mixture. (Bottom) Extracted Ion Chromatograms (XICs) of the calibrant ions from 6-Peptide Mixture generated using XIC Manager in PeakView™ Software 1.1.

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Acquisition The acquisition method for intact protein analysis typically includes only a single TOF-MS experiment with mass range being the only parameter that may vary with sample. Below are guidelines for MS parameters necessary to build an acquisition method for intact proteins on the TripleTOF™ 5600 System.

• Mass Range: This will vary depending on the details of the sample, but m/z 500 –3000 is an adequate range for most intact protein applications. For optimum sensitivity, it is best to avoid using a wider window than is necessary at either the high- or low-m/z end of the range. Additionally for optimum mass accuracy, it is important that the upper limit of the mass range be identical for the calibration and sample acquisition methods.

• Accumulation Time: Chromatographic peaks for intact proteins are generally several seconds wide so an accumulation time of 1s is sufficient.

• Source/Gas Tab: Optimum DuoSpray™ source parameters will vary depending on flow rate. For 200 µL/min flow rate, GS1=45, GS2=45 and a ISV=5200 V are typical settings. Source temperature will also vary with flow rate, but 500°C is a good starting point.

• Compound Tab: The optimum transmission / declustering potential for proteins occurs at DP = 80 – 120V. Collision energy (CE) is maintained at the default of 10V. The collision gas (CAD) can also be left at the default (6) although for larger proteins (>50 kDa) there may be some benefit to

increasing the CAD gas setting to 8 – 10. This is to provide additional cooling of large proteins in the collision cell prior to entering the time-of-flight chamber.

• Q1 Transmission Window: Once mass range has been set in TOF MS mode, allow the Q1 Transmission Window to be selected automatically by checking ‘Auto Adjust with mass’ or by clicking ‘Suggest’.

• Time Bins to Sum: This parameter determines the number of data points per m/z unit (and consequently points across each spectral peak) in the mass spectrum. For smaller proteins which yield isotopic resolution (approx. <20 kDa), the default value of 4 is used for this parameter. For larger proteins, signal-to-noise can be improved without sacrificing resolution or mass accuracy by using a ‘Time Bins to Sum’ value of 40-80.

There are multiple chromatography solutions to choose from for intact protein analysis. Most use a form of C4 or C8 reversed-phase technology running in the 200 – 500 µL/min flow rate range (Figure 5). Mobile phases are usually comprised of water / acetonitrile / formic acid, although for some applications it is recommended that 2-propanol be added as a supplemental modifier to the organic mobile phase. All intact protein LC solutions are compatible with the TripleTOF™ 5600 System.

Prior to submission of a batch for analysis, it is recommended that Intact Protein Mode be engaged from the Scripts menu2. This utility optimizes acquisition and signal processing algorithms specifically for the challenges presented by intact protein analysis. The Intact Protein script (Figure 4) also allows the user to automatically decrease the MCP voltage by 100V. Counter-intuitively, this often improves sensitivity for proteins by significantly reducing signal resulting from singly charged chemical background, while having little or no effect on the signal resulting from multiply charge protein ions. Changes made by the Intact Protein script remain in effect until Intact Protein Mode is turned off.

Figure 5. LC/MS Analysis of Intact Proteins. Best intact protein results are often obtained by performing a reverse phase LC (top) before MS analysis, to desalt and clean up the protein sample.

Figure 4: Intact Protein Mode Script. Intact Protein Mode optimizes acquisition and signal processing algorithms for intact protein analysis. The checkbox allows automatic reduction of detector voltage by 100 V to improve signal-to-noise for multiply charged protein peaks. It is recommended to engage Intact Protein Mode for all protein analysis.

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Data Processing For intact mass determination of protein mass spectra (calculation of a single molecular weight from a distribution of multiple charge states), BioAnalyst™ Software contains a number of useful tools.

Manual reconstruction: This simple tool enables the calculation of an intact protein mass from multiple user-selected peaks. Multiple charge states or multiple isotope peaks can be used for the calculation.

Bayesian Protein Reconstruction: This algorithm enables the automated deconvolution and reconstruction of mixed protein spectrum. First the data is baselined. The user then has the option to sharpen the peaks by deconvolution. This optional deconvolution consists of picking an observed peak that best represents the data, modeling that peak, and then using that model to deconvolve all peaks in the spectrum. Then a maximum entropy algorithm is used for mass reconstruction, creating a mass graph. Peak detection is then performed on the mass graph to obtain the MW of the detected protein species. Details of the capabilities and instructions for use of BioAnalyst TF are included in the Using BioAnalyst TF Tutorial3.

Figure 7: Confirmation of Charge State and Isotope Information. If the protein mass is known, the intact protein MS data can be processed using PeakView™ Software. Upper pane shows spectrum for horse myoglobin with overlays of the theoretical charge states as computed by the Hypermass calculator. Middle pane shows the multi-function mass calculator tool. Lower pane shows the 20+ charge state of myoglobin with theoretical isotope ratios overlaid.

Figure 6. Mass Reconstruction of Intact Protein MS Data. To determine the exact mass from the protein MS data, the reconstruction tools in BioAnalyst™ Software can be used. First, peak deconvolution can be performed (top) followed by reconstruction, which produces both a mass table (middle) and a mass graph (bottom).

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If the mass of the protein is known or has been determined using mass reconstruction tools, PeakView™ Software provides easy-to-use calculations for sequence mass, elemental composition, mass accuracy, theoretical isotope ratios, and Hypermass. The Hypermass tool can be used to compare protein spectra to theoretical charge state distributions (Figure 7) to check the fidelity of the MS results.

Summary The TripleTOF™ 5600 System is a powerful solution for performing intact protein analysis. It combines high resolution and mass accuracy with high sensitivity and wide mass range. LC AutoCalibration provides easy, automated calibration to ensure long-term mass accuracy throughout extended sample batches. Use of a basic TOF-MS method with the parameters described above allows for easy method generation. Finally, the Intact Protein Mode tool enables the user to quickly optimize acquisition and signal processing for best protein detection.

References 1. LCMS Protein / Peptide Mass Standards Kit - P/N 4368642.

Exact masses for the individual peptides can be found in the kit documentation.

2. The Intact Protein Mode script can be found at C:\Program Files\Analyst\Scripts\IntactProteinMode. Navigate to this directory and run Setup.exe to install the script. A window will appear to indicate successful installation when it is complete. The script will then be accessible in the Scripts menu in Analyst® Software TF 1.5.1.

3. Using BioAnalyst™ Software Tutorial – available upon installation of the software.

For Research Use Only. Not for use in diagnostic procedures.

© 2011 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.

Publication number: 3240911-01