Forced degradation analysis of recombinant adeno-associated virus serotype 8 based on analytical anion exchange chromatography coupled to orthogonal characterization

Oligonucleotide and mRNA Analysis Resource Roundup

NIST26 introduces integrated chromatogram processing, deconvolution, and library searching for EI GC-MS and LC-MS/MS data, but the workflow begins with one essential requirement: chromatographic data must be available in the mzML format. In his presentation <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/pdf/Conversion_to_mz_ML_63e134bb34.pdf">Conversion to mzML Format for NIST26 Chromatogram Processing</a>, James Little of Mass Spec Interpretation Services explains how vendor data can be converted to mzML using either instrument-vendor software or the freely available MSConvert tool from ProteoWizard, and highlights several settings that are critical for successful processing in NIST26.<h4>NIST26 Chromatogram processing requires mzML</h4>For the chromatogram-processing workflow described in the presentation, mzML is the accepted input format. Proprietary LC-MS or GC-MS data therefore need to be converted before they can be loaded into the NIST26 chromatogram-processing environment.There are two principal ways to obtain an mzML file:<ul><li>conversion using software supplied by the instrument manufacturer,</li><li>conversion using MSConvert, which is included in the free ProteoWizard software package.</li></ul>Using an open format such as mzML also provides a practical way to move mass spectrometry data between different software environments without relying exclusively on proprietary vendor formats.<h4>Using ProteoWizard and MSConvert</h4>MSConvert is installed as part of the complete ProteoWizard package rather than as a separate application. After installing ProteoWizard, users can access either the graphical MSConvert interface or the command-line version.The presentation recommends the stable release for routine work, while noting that newer or nightly builds may sometimes provide improved support for recently introduced vendor formats or additional control over data-processing filters. This can be particularly relevant when handling vendor files in which centroiding behavior or peak picking requires closer attention.For most users, the graphical interface provides a straightforward route from proprietary raw data to an mzML file suitable for further NIST26 processing.<h4>Start by selecting mzML as the output format</h4>The first step in MSConvert is to select the source file or dataset using the Browse function.The software supports a broad range of mass spectrometry data formats, and the file-selection window can either be restricted to a particular vendor format or set to display any supported spectral format.After selecting the source data, the output format must be set to mzML. The screenshots in the presentation show this setting directly in the MSConvert options panel before the conversion is started.The user can also select the destination folder and other file-output options before adding the selected dataset to the conversion queue.<h4>Add the data and start the conversion</h4>Once the source file and output settings have been selected, the file is added to the conversion list using the Add command.The conversion itself is then launched with Start.For simple datasets that are already stored in the correct spectral representation, this may be all that is required. However, James Little emphasizes an additional requirement that is particularly important for NIST26: the data must be centroided.<h4>Centroided data are required</h4>According to the presentation, profile-mode data are not acceptable for this NIST26 chromatogram-processing workflow. The spectra must instead be in centroided, or peak-picked, form.Profile spectra contain the full measured shape of each mass spectral signal across the m/z axis. Centroided data reduce these profiles to discrete peaks represented by their characteristic m/z and intensity values.If the original vendor data are still in profile mode, centroiding can be performed during conversion by adding the Peak Picking filter in MSConvert.This means that the conversion step can perform two operations at once:vendor raw data → centroiding / peak picking → mzMLThe resulting mzML file can then be used as input for NIST26 chromatogram processing.<h4>Peak Picking must be the first filter</h4>One of the most important practical details in the presentation is the position of the Peak Picking operation in the MSConvert filter sequence.If centroiding is required, Peak Picking must be the first algorithm or filter in the list. Placing another processing function before it can interfere with the intended conversion workflow.MSConvert allows the user to select the Peak Picking filter, define the relevant MS levels, and add it to the processing sequence before starting conversion. The example shown in the presentation applies vendor-based peak picking to selected MS levels.If there is uncertainty about whether the source data are already centroided, the Peak Picking filter can still be included. According to the presentation, applying it to data that are already centroided does not alter the file unnecessarily.This provides a useful safeguard when the acquisition mode of an older dataset is uncertain.<h4>Selecting the appropriate MS levels</h4>Peak picking does not necessarily have to be applied identically to every spectrum in a dataset. MSConvert allows users to specify the MS levels to which the filter should be applied.This is especially relevant for LC-MS/MS data, where a file can contain both precursor-level MS spectra and tandem MS spectra.The conversion settings should therefore reflect the way the original data were acquired and how those data will subsequently be processed in NIST26.<h4>Be aware of changes in file size</h4>The presentation also notes that converted files may become substantially different in size from the original vendor files.This is worth considering when working with large LC-MS/MS datasets or processing batches of chromatograms. The size of the resulting mzML file can depend on the original acquisition mode, centroiding, compression settings, the number of MS levels, and other output parameters.Storage requirements should therefore be considered before converting large collections of raw data.<h4>A simple workflow for preparing data for NIST26</h4>The overall procedure presented by James Little can be summarized as a short sequence:<ol><li>Obtain the original vendor LC-MS or GC-MS data.</li><li>Open the data in MSConvert or use the vendor's own conversion software.</li><li>Select mzML as the output format.</li><li>Check whether the spectra are centroided.</li><li>If necessary, add Peak Picking as the first processing filter.</li><li>Select the appropriate MS levels.</li><li>Add the file to the conversion list.</li><li>Start the conversion.</li><li>Use the resulting mzML file for NIST26 chromatogram processing.</li></ol>The procedure itself is straightforward, but the centroiding requirement is important. A file can have the correct <code>.mzML</code> extension and still be unsuitable for the workflow if its spectra remain in profile mode.<h4>From vendor data to integrated NIST26 processing</h4>File conversion may appear to be a relatively minor step compared with deconvolution, spectral interpretation, and library searching, but it establishes the foundation for the entire downstream workflow.By using mzML as the common input format, NIST26 can process data originating from different instrument platforms through a more consistent workflow. ProteoWizard and MSConvert provide a practical bridge between proprietary mass spectrometry files and this standardized format.The key message from the presentation is therefore simple: convert to mzML, ensure the spectra are centroided, and place Peak Picking first whenever centroiding is required. Once those conditions are met, the data are ready for the integrated chromatogram-processing and library-searching capabilities available in NIST26.

Conversion to mzML Format for NIST26 Chromatogram Processing

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Phenomenex Strata Eco Screen Solid Phase Extraction (SPE)

This proprietary normal phase sorbent is topped with sodium sulfate to remove any excess water and is used for the extraction of hydrocarbons from environmental samples.

Specialty Solid Phase Extraction (SPE) SorbentThis proprietary normal phase sorbent is topped with sodium sulfate to remove any excess water and is used for the extraction of hydrocarbons from environmental samples, resulting in high recoveries of naphthalene.

Phenomenex Strata EPH Solid Phase Extraction (SPE)

This specialty normal phase sorbent was developed for the fractionation of aliphatic and aromatic hydrocarbons from environmental samples.

Specialty Solid Phase Extraction (SPE) SorbentThis specialty normal phase sorbent was developed for the fractionation of aliphatic and aromatic hydrocarbons from environmental samples.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Strata_eph_6f99720a51.webp" alt="Phenomenex: Strata-EPH" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Strata_eph_6f99720a51.webp 245w," sizes="100vw" width="500" height="150"><ul><li>Surface Area (m²/g): -</li><li>Separation Mode: Multi-mode</li><li>Particle Size: 200</li><li>Recommended Use: Fractionation of aliphatic and aromatic hydrocarbons from environmental samples</li></ul><h4>Starting Method</h4><h5>Strata EPH Normal Phase</h5><ul><li>Condition: IPA/DCM</li><li>Equilibrate: Hexane</li><li>Load: Pretreated sample</li><li>Wash: 5 % DCM in Hexane</li><li>Elute: 1:1 Hexane / DCM or 1:1 Hexane / IPA</li></ul>Based on 100 mg/1 mL sorbent mass. The above is a convenient starting point for SPE method development, further optimization may be required to tailor the method for your specific extraction needs.

Phenomenex Strata PAH Solid Phase Extraction (SPE)

Strata PAH is a proprietary sorbent designed to provide high recoveries of polycyclic aromatic hydrocarbons from water.

Specialty Solid Phase Extraction (SPE) SorbentStrata PAH (Polycyclic Aromatic Hydrocarbons) is a proprietary sorbent designed to provide high recoveries of polycyclic aromatic hydrocarbons from water (as specified in EPA Method 550.1) while simultaneously removing humic acids from the extract.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Strata_pah_c48e1fab83.webp" alt="Phenomenex: Strata-PAH" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Strata_pah_c48e1fab83.webp 245w," sizes="100vw" width="500" height="150"><ul><li>Surface Area (m²/g): -</li><li>Separation Mode: Reversed Phase</li><li>Particle Size: 55</li><li>Recommended Use: Extraction of polycyclic aromatic hydrocarbons from water (as specified in EPA Method 550.1) while simultaneously removing humic acids from the extract</li></ul>

Phenomenex Strata PFAS Solid Phase Extraction (SPE)

A new stacked cartridge able to offer 2x gains in productivity and reduce the need for multiple tubes, transfer steps, and wasted time. 

Specialty Solid Phase Extraction (SPE) SorbentA new stacked cartridge able to offer 2x gains in productivity and reduce the need for multiple tubes, transfer steps, and wasted time. This product is recommended to be used for EPA 1633 and DOD QSM 5.1/5.3 guidelines to minimize multiple sample but still provide high recoveries of low level PFAS (Per- and Polyfluoroalkyl Substances) in solid samples, soils, biota, sediments, or non-drinking water samples.<ul><li>Surface Area (m²/g): -</li><li>Separation Mode: Multi-Mode</li><li>Pore Size: 0</li><li>Recommended Use: for EPA 1633 and DOD QSM 5.1/5.3 applications for diverse matrices and non-drinking water PFAS applications</li></ul><h4>Stacked SPE Cartridges for Efficient PFAS Extraction</h4>Strata PFAS is a stacked solid phase extraction cartridge for EPA method 1633 and DOD QSM 5.1/5.3 applications.<ul><li>Reliable SPE with 2x gains in productivity</li><li>Reproducible extraction</li><li>Reduced need for multiple tubes, transfer steps, and wasted time</li></ul>

Phenomenex Strata CN Solid Phase Extraction (SPE)

Strata CN is a polar phase with slight hydrophobic selectivity in reversed phase mode and moderate polar selectivity in normal phase mode.

Normal Phase Solid Phase Extraction (SPE)A polar phase with slight hydrophobic selectivity in reversed phase mode and moderate polar selectivity in normal phase mode.<figure class="image"><img style="aspect-ratio:500/150;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Strata_cn_769c96c476.webp" alt="Phenomenex: Strata-CN" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Strata_cn_769c96c476.webp 245w," sizes="100vw" width="500" height="150"></figure><ul><li>Surface Area (m²/g): -</li><li>Separation Mode: Normal Phase</li><li>Particle Size: 55</li><li>Recommended Use: Extraction of polar compounds such as those that contain hydroxyl groups.</li></ul><h4>Starting Method</h4><ul><li>Condition: IPA/DCM</li><li>Equilibrate: Hexane</li><li>Load: Pretreated sample</li><li>Wash: 5 % DCM in Hexane</li><li>Elute: 1:1 Hexane / DCM or 1:1 Hexane / IPA</li></ul>Based on 100 mg/1 mL sorbent mass. The above is a convenient starting point for SPE method development, further optimization may be required to tailor the method for your specific extraction needs.

Phenomenex Strata Silica (Si-1) Solid Phase Extraction (SPE)

Strata Silica (Si-1) is a normal phase SPE sorbent with unbonded silica particle that offers strong polar selectivity.

Normal Phase Solid Phase Extraction (SPE)A polar phase with slight hydrophobic selectivity in reversed phase mode and moderate polar selectivity in normal phase mode.<figure class="image"><img style="aspect-ratio:500/150;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Strata_si_e4eb885006.webp" alt="Phenomenex: Strata-Silica (Si-1)" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Strata_si_e4eb885006.webp 245w," sizes="100vw" width="500" height="150"></figure><ul><li>Surface Area (m²/g): 550, m²/g, null m²/g, 490 m²/g</li><li>Separation Mode: Normal Phase</li><li>Pore Size: 70</li><li>Particle Size: 55</li><li>Recommended Use: Extraction of polar compounds that are similar in structure</li></ul><h4>Starting Method</h4><h5>Strata Normal Phase Method</h5><ul><li>Condition: IPA/DCM</li><li>Equilibrate: Hexane</li><li>Load: Pretreated sample</li><li>Wash: 5 % DCM in Hexane</li><li>Elute: 1:1 Hexane / DCM or 1:1 Hexane / IPA</li></ul>Based on 100 mg/1 mL sorbent mass. The above is a convenient starting point for SPE method development, further optimization may be required to tailor the method for your specific extraction needs.

Phenomenex Strata Melamine Solid Phase Extraction (SPE)

A proprietary phase that allows for the simultaneous extraction of melamine and cyanuric acid out of food and biological samples.

Specialty Solid Phase Extraction (SPE) SorbentA proprietary phase that allows for the simultaneous extraction of melamine and cyanuric acid out of food and biological samples.

Phenomenex Strata SCX Solid Phase Extraction (SPE)

A benzene sulfonic acid group is bonded to the surface of the silica particle, giving strong cation-exchange selectivity.

Ion-Exchange Solid Phase Extraction (SPE) SorbentA benzene sulfonic acid group is bonded to the surface of the silica particle, giving strong cation-exchange selectivity.<ul><li>Extraction of 1 °, 2 °, and 3 amines</li></ul><figure class="image"><img style="aspect-ratio:791/384;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Strata_SCX_SPE_f35df1035c.jpg" alt="Phenomenex: Strata SCX" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Strata_SCX_SPE_f35df1035c.jpg 245w," sizes="100vw" width="791" height="384"></figure><ul><li>Surface Area (m²/g): -</li><li>Separation Mode: Ion Exchange</li><li>Particle Size: 55</li><li>Recommended Use: Extraction of weakly basic compounds</li></ul><h4>Starting Method</h4><h5>Strata SCX – Strong Cation-Exchange</h5><ul><li>Condition: 1 mL Methanol</li><li>Equilibrate: 1 mL Acidified Water</li><li>Load: Pretreated sample (acidified)</li><li>Wash 1: 1 mL 0.1N HCl in Water</li><li>Wash 2: 1 mL 0.1N HCl in Methanol, dry under vacuum for 2-5 min</li><li>Elute: 1 mL 5 % NH4OH in Methanol</li></ul>Based on 100 mg/1 mL sorbent mass. The above is a convenient starting point for SPE method development, further optimization may be required to tailor the method for your specific extraction needs.

Phenomenex Strata NH2 Solid Phase Extraction (SPE)

This amino phase offers strong polar selectivity and hydrogen bonding under normal phase conditions or can be used as a weak anion-exchange sorbent.

Normal Phase Solid Phase Extraction (SPE)This amino phase offers strong polar selectivity and hydrogen bonding under normal phase conditions or can be used as a weak anion-exchange sorbent.<figure class="image"><img style="aspect-ratio:500/150;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Strata_nh2_a7c3599f55.webp" alt="Phenomenex: Strata-NH2" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Strata_nh2_a7c3599f55.webp 245w," sizes="100vw" width="500" height="150"></figure><ul><li>Surface Area (m²/g): -</li><li>Separation Mode: Normal Phase</li><li>Pore Size: 100</li><li>Particle Size: 25, 55</li><li>Recommended Use: Strong polar selectivity and hydrogen bonding under normal phase conditions or can be used as a weak anion-exchange sorbent</li></ul><h4>Starting Method</h4><h5>Strata NH2 – Weak Anion-Exchange</h5><ul><li>Condition: 1 mL Methanol</li><li>Equilibrate: 1 mL Water, pH 6-7</li><li>Load: Pretreated sample, pH 6-7</li><li>Wash 1: 1 mL 25mM Ammonium Acetate Buffer, pH 6-7</li><li>Wash 2: 1 mL Methanol, dry under vacuum for 2-5 min</li><li>Elute Any Acid: 1 mL 5% NH4OH in Methanol</li><li>Elute Weak Acids: 1 mL 5% Formic Acid in Methanol</li></ul>Based on 100 mg/1 mL sorbent mass. The above is a convenient starting point for SPE method development, further optimization may be required to tailor the method for your specific extraction needs.

Phenomenex Strata SAX Solid Phase Extraction (SPE)

The quaternary amine group on this sorbent remains positively charged under all conditions, giving a strong anion-exchange mechanism of retention.

Ion-Exchange Solid Phase Extraction Sorbent (SPE)The quaternary amine group on this sorbent remains positively charged under all conditions, giving a strong anion-exchange mechanism of retention.<figure class="image"><img style="aspect-ratio:500/150;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Strata_sax_470d923b27.webp" alt="Phenomenex: Strata-SAX" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Strata_sax_470d923b27.webp 245w," sizes="100vw" width="500" height="150"></figure><ul><li>Surface Area (m²/g): -</li><li>Separation Mode: Ion Exchange</li><li>Particle Size: 55</li><li>Recommended Use: Extraction of weakly acidic compounds</li></ul><h4>Starting Method</h4><h5>Strata SAX – Strong Anion-Exchange</h5><ul><li>Condition: 1 mL Methanol</li><li>Equilibrate: 1 mL DI Water</li><li>Load: Pretreated sample, pH 6-7</li><li>Wash 1: 1 mL 25mM Ammonium Acetate Buffer, pH 6-7</li><li>Wash 2: 1 mL Methanol, dry under vacuum for 2-5 min</li><li>Elute: 1 mL 5% Formic Acid in Methanol</li></ul>Based on 100 mg/1 mL sorbent mass. The above is a convenient starting point for SPE method development, further optimization may be required to tailor the method for your specific extraction needs.

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Conversion to mzML Format for NIST26 Chromatogram Processing

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