Rapid characterization of adeno-associated virus (AAV) capsid proteins using microchip ZipChip CE-MS

Adeno-associated viruses (AAVs) are viral vectors used as delivery systems for gene therapies. Intact protein characterization of AAV viral capsid proteins (VPs) and their post-translational modifications is critical to ensuring product quality. In this study, microchip-based ZipChip capillary electrophoresis-mass spectrometry (CE-MS) was applied for the rapid characterization of AAV intact VPs, specifically full and empty viral capsids of serotypes AAV6, AAV8 and AAV9, which was accomplished using 5 min of analysis time. Low levels of dimethyl sulfoxide (4%) in the background electrolyte (BGE) improved MS signal quality and component detection. A sensitivity evaluation revealed consistent detection of VP proteoforms when as little as 2.64 × 106 viral particles (≈26.4 picograms) were injected. Besides the traditional VP proteoforms used for serotype identification, multiple VP3 variants were detected, including truncated VP3 variants most likely generated by leaky scanning as well as unacetylated and un-cleaved VP3 proteoforms. Phosphorylation, known to impact AAV transduction efficiency, was also seen in all serotypes analysed. Additionally, low abundant fragments originating from either N- or C-terminus truncation were detected. As the aforementioned VP components can impact product quality and efficacy, the ZipChip’s ability to rapidly characterize them illustrates its strength in monitoring product quality during AAV production. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-023-05097-5.


Table S1
BPF 5.1 software parameter settings for intact mass analysis Table S2 BPF 5.1 software parameter settings for peptide mapping analysis

Table S3
Effect of sample conditions on MS intensity and AAV8 viral capsid protein (VP) charge state

Table S4
Detected AAV8 VPs under investigated sample analysis conditions

Table S5
Empty AAV8 capsid dilution series for ZipChip limit of detection (LoD) evaluation

Table S6
Unknown components detected in the empty and full capsids of AAV6, AAV8, and AAV9

Table S7
Select peptides found in both empty and full AAV6 peptide mapping analysis to confirm presence of VPs and fragments detected during intact mass analysis.Tandem MS (MS/MS) spectra of the highlighted peptides is shown in Figures S2-S7 as illustrative examples of the quality of the peptides identified for each VP or fragment.Peptide information displayed is from the analysis of the full AAV6 capsids

Table S8
Select peptides found in both empty and full AAV8 peptide mapping analysis to confirm presence of VPs and fragments detected during intact mass analysis.MS/MS spectra of the highlighted peptides is shown in Figures S8-S14 as illustrative examples of the quality of the peptides identified for each VP or fragment.Peptide information displayed is from the analysis of the full AAV8 capsids

Table S9
Select peptides found in both empty and full AAV9 peptide mapping analysis to confirm presence of VPs and fragments detected during intact mass analysis.MS/MS spectra of the highlighted peptides is shown in Figures S15-S26 as illustrative examples of the quality of the peptides identified for each VP or fragment.Peptide information displayed is from the analysis of the full AAV9 capsids

Table S10
Acetylation and phosphorylation PTMs identified during peptide mapping of AAV6 empty and full capsids with a relative abundance ≥ 1%.~ indicates that the exact location of the modification could not be determined by BPF 5.1, but the modification is near or on the listed residue

Table S11
Acetylation and phosphorylation PTMs identified during peptide mapping of AAV8 empty and full capsids with a relative abundance ≥ 1%.~ indicates that the exact location of the modification could not be determined by BPF 5.1, but the modification is near or on the listed residue

Table S12
Acetylation and phosphorylation PTMs identified during peptide mapping of AAV9 empty and full capsids with a relative abundance ≥ 1%.~ indicates that the exact location of the modification could not be determined by BPF 5.1, but the modification is near or on the listed residue

Figure S1
Plot of total ion electropherogram area (blue line on top) and extracted apex MS signal intensity (orange line on bottom) versus Vps injected during LoD study

Figure S2
Tandem MS (MS/MS) spectra of the AADGYLPDWLEDNLSEGIRE (A2-E21) peptide, with acetylation at A2, from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP1 capsid protein identified during intact mass analysis (Table S7)

Figure S3
MS/MS spectra of the APGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGD (A139-D178) peptide from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.
Peptide is an example of the detected peptides that indicate the presence of the VP2 capsid protein identified during intact mass analysis (Table S7)

Figure S4
MS/MS spectra of the ASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITT (A204-T242) peptide, with acetylation at A204, from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the VP3 capsid protein identified during intact mass analysis (Table S7)

Figure S5
MS/MS spectra of the ADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRT (A212-T246) peptide, with acetylation at A212, from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3 Variant capsid protein identified during intact mass analysis (Table S7)

Figure S6
MS/MS spectra of the RAVFQAKKRVLEPFGL (R116-L131) peptide from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the R116-VP1 fragment identified during intact mass analysis (Table S7)

Figure S7
MS/MS spectra of the EEIKATNPVATERFGTVAVNLQSSSTD (E564-D590) peptide from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-D590 fragment identified during intact mass analysis (Table S7)

Figure S8
MS/MS spectra of the AADGYLPDWLEDNLSEGIRE (A2-E21) peptide, with acetylation at A2, from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP1 capsid protein identified during intact mass analysis (Table S8)

Figure S9
MS/MS spectra of the APGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPD (A139-D185) peptide, with phosphorylation around S149 (exact location could not be determined), from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.
Peptide is an example of the detected peptides that indicate the presence of the VP2 capsid protein identified during intact mass analysis (Table S8).The relatively poor spectrum is a result of the phosphorylation present which impacts digestion efficiency of the pepsin, resulting in the large peptide identified

Figure S10
MS/MS spectra of the AAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITT (A205-T243) peptide, with acetylation at A205, from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3 capsid protein identified during intact mass analysis (Table S8)

Figure S11
MS/MS spectra of the AAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVIT (A205-T242) peptide, with no acetylation, from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the VP3 capsid protein without N-term acetylation identified during intact mass analysis (Table S8)

Figure S12
MS/MS spectra of the ADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRT (A213-T247) peptide, with acetylation at A213, from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3 Variant capsid protein identified during intact mass analysis (Table S8)

Figure S13
MS/MS spectra of the VEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPD (V132-D185) peptide, with phosphorylation around S149 (exact location could not be determined), from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the V132-VP1 fragment identified during intact mass analysis (Table S8).The relatively poor spectrum is a result of the phosphorylation present which impacts digestion efficiency of the pepsin, resulting in the large peptide identified

Figure S14
MS/MS spectra of the GAPMADNNEGADGVGSSSGNWHCDSTWLGDRVIT (G209-T242) peptide from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the G209-VP3 fragment identified during intact mass analysis (Table S8)

Figure S15
MS/MS spectra of the AADGYLPDWLEDNLSEGIRE (A2-E21) peptide, with acetylation at A2, from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP1 capsid protein identified during intact mass analysis (Table S9)

Figure S16
MS/MS spectra of the APGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTM (A139-M203) peptide, from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the VP2 capsid protein identified during intact mass analysis (Table S9)

Figure S17
MS/MS spectra of the ASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVIT (A204-T241) peptide, with acetylation at A204, from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3 capsid protein identified during intact mass analysis (Table S9)

Figure S18
MS/MS spectra of the MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRT (M203-T246) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the M203-VP3 capsid protein identified during intact mass analysis (Table S9).This is a VP3 protein that didn't undergo expected N-term methionine cleavage and subsequent Nterm acetylation

Figure S19
MS/MS spectra of the RAVFQAKKRLLEPLGL (R116-L131) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the R116-VP1 fragment identified during intact mass analysis (Table S9)

Figure S20
MS/MS spectra of the LVEEAAKTAPGKKRPVE (L131-E147) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the L131-VP1 fragment identified during intact mass analysis (Table S9)

Figure S21
MS/MS spectra of the FGQTGDTESVPDPQPIGEPPAAPSGVGSL (F173-L201) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the F173-VP2 fragment identified during intact mass analysis (Table S9)

Figure S22
MS/MS spectra of the IKNTPVPAD (I649-D657) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-D657 fragment identified during intact mass analysis (Table S9)

Figure S23
MS/MS spectra of the ALNGRNSLMNPGPAMASHKEGEDRFFPLS (A510-S538) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-S538 fragment identified during intact mass analysis (Table S9)

Figure S24
MS/MS spectra of the FAWPGASSWALNGRNSLM (F501-M518) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-M518 fragment identified during intact mass analysis (Table S9)

Figure S25
MS/MS spectra of the AVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALN (A472-N512) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.
Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-N512 fragment identified during intact mass analysis (Table S9)

Figure S26
MS/MS spectra of the YLYYLS (Y443-S448) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-S448 fragment identified during intact mass analysis (Table S9) Supplementary Information 1: Sample preparation for peptide mapping digestion of AAV capsids Sample preparation for AAV peptide mapping digestion was performed as follows.For each digestion, 4 µg of AAV sample was brought to a total volume of 50 µL with LC-MS grade water in a 500 µL Eppendorf protein LoBind tube (Eppendorf, Dublin).148 µL of SMART Digest buffer and 2 µL of 0.5 M TCEP (5 mM final concentration) were then added for reduction and denaturation.Next 15 µL of pepsin beads were combined with 100 µL SMART Digest buffer and a digest wash solution composed of 50 µL of SMART Digest buffer and 150 µL of LC-MS grade water was prepared.The denatured AAV sample, the pepsin bead mixture, and the wash solution were then added to different wells of a 96deepwell plate (Thermo Scientific, Vantaa, Finland).Sample digestion on the pepsin beads was performed using a Thermo Scientific KingFisher Duo Prime purification system under the control of Thermo Scientific BindIt software, version 4.0.Samples AAV6 Empty (AAV6E), AAV6 Full (AAV6F), AAV8 Empty (AAV8E), AAV9 Empty (AAV9E), and AAV9 Full (AAV9F) were incubated for 30 minutes while AAV8 Full (AAV8F) was incubated for 45 minutes.All incubations were performed at 70°C, set to medium mixing speed.After incubation, the beads were removed, and remaining sample volume was transferred to 1.5 mL Eppendorf protein LoBind tubes (Eppendorf, Dublin).Samples were centrifuged at 14,000 g for 5 min to pellet any pepsin beads that had not been removed.The remaining supernatant was then transferred to a vial for liquid chromatography-mass spectrometry (LC-MS) for analysis.

Supplementary Information 2: Detailed AAV peptide mapping LC-MS analysis parameters
Peptide mapping LC-MS analysis was performed in technical triplicate using a Vanquish Neo UHPLC coupled to an Orbitrap Exploris 480 (Thermo Scientific, Bremen, Germany) following a modified version of the procedure described in Guapo et al (9).Mobile phase A was UHPLC-MS grade water containing 0.1 % (v/v) FA and mobile phase B was Optima™ LC-MS grade ACN containing 0.1 % (v/v) FA.Per injection, 250ng of digested AAV sample from Section 2.3 (assumed 100% digestion) was separated using a Thermo Scientific Easy-Spray™ PepMap™ Neo C18 column, 2 µm, 75 µm × 50 cm (Thermo Scientific, Sunnyvale, CA, USA).After injection, a hold at 2% B was performed for 10 min before peptide separation was performed using a linear gradient (Curve 5) from 2% B to 25% B over 60 min, followed by an increase to 45% B over 30 min.A column wash was then performed over 10 min by performing a step gradient to 80% B followed by an isocratic hold for 5 min.A step gradient to 2% B followed by a hold for 2 min was performed before a step gradient back to 80% B with a subsequent hold for 3 min was set to complete the wash.The flow rate was kept constant at 250 nL/min and the column temperature was maintained at 45°C.
Data-dependent acquisition (DDA)-MS analysis was performed in positive ion mode.Full scans were acquired at a resolution of 60,000 between a mass range of m/z 200-2,000.The RF Lens was set to 40% while the AGC target was set to 1.0 × 10 6 with a maximum injection time of 50ms and 1 microscan.MS-MS (MS 2 ) fragment scans were acquired using a resolution setting of 15,000 with an AGC target of 4.5 × 10 4 , a maximum injection time of 70 ms, an isolation window of m/z 1.2, and a signal intensity threshold of 5.0 × 10 3 .Fragmentation of the twenty most abundant precursor ions was performed using a normalized collision energy set to 28 with a dynamic exclusion set for 45s.Charge states from 2-7 were included for detection.The MS tune parameters were as follows: Spray voltage was set to 1.7 kV; Capillary temperature was set to 300°C.

Supplementary Information 3: Detailed AAV peptide mapping data analysis parameters
Peptide identification and relative post translational modification (PTM) quantitation was performed using a modified version of what is outlined by Guapo et al (9).Briefly, a peptide mapping analysis experiment was created in BioPharma Finder™ (BPF) Version 5.1 (Thermo Scientific, San Jose, CA, USA) to process the raw data files generated from Supplementary Information 1 using the parameters summarized in Table S2.Only peptides with a ≥ 95% confidence score, ≥ 1 x 10 5 average MS area, and within ± 5 ppm mass accuracy were included for sequence coverage evaluation.Peptides with adducts, unknown modifications, gas phase ions, and non-specific generated peptides fitting these parameters were filtered from the results and thus not utilized for sequence coverage determination.
For PTM quantitation the same parameters as for peptide sequence coverage were applied.PTMs were identified automatically in BPF by their mass differences compared to unmodified peptides generated from pepsin digestion.Relative PTM abundance was expressed in BPF as a percentage of PTM presence on a peptide to all forms of said peptide present.Manual validation of peptides selected for relative PTM quantitation was performed in addition to automatic software assignment to ensure accurate quantitation.A213(Ac)-VP3 Variant 3.15 × 10 8 1.48 × 10 9 1.43 × 10 9 35.25 22.40 20.20 39.50 42.20 42.80 75.25 63.60 62.00 *This is an average of the data obtained from processing the 5 injections run for each condition in BPF 5.1.VP = viral protein, Ac = N-Term Acetylation, P = Phosphorylated, -= Not identified under that condition        S3 MS/MS spectra of the APGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGD (A139-D178) peptide from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the VP2 capsid protein identified during intact mass analysis (Table S7)

Relative Abundance
Figure S4 MS/MS spectra of the ASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITT (A204-T242) peptide, with acetylation at A204, from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the VP3 capsid protein identified during intact mass analysis (Table S7 (Ac)VP3 A204(Ac)-T242

Relative Abundance
Figure S5 MS/MS spectra of the ADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRT (A212-T246) peptide, with acetylation at A212, from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3 Variant capsid protein identified during intact mass analysis (Table S7)

1045.8214
Figure S6 MS/MS spectra of the RAVFQAKKRVLEPFGL (R116-L131) peptide from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the R116-VP1 fragment identified during intact mass analysis (Table S7)

Relative Abundance
Figure S7 MS/MS spectra of the EEIKATNPVATERFGTVAVNLQSSSTD (E564-D590) peptide from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-D590 fragment identified during intact mass analysis (Table S7) Figure S8 MS/MS spectra of the AADGYLPDWLEDNLSEGIRE (A2-E21) peptide, with acetylation at A2, from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.

496
Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP1 capsid protein identified during intact mass analysis (Table S8)

(Ac)VP1 A2(Ac)-E21
Figure S9 MS/MS spectra of the APGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPD (A139-D185) peptide, with phosphorylation around S149 (exact location could not be determined), from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the VP2 capsid protein identified during intact mass analysis (Table S8).The relatively poor spectrum is a result of the phosphorylation present which impacts digestion efficiency of the pepsin, resulting in the large peptide identified

928.4889
Figure S10 MS/MS spectra of the AAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITT (A205-T243) peptide, with acetylation at A205, from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3 capsid protein identified during intact mass analysis (Table S8

2280.0303
Figure S11 MS/MS spectra of the AAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVIT (A205-T242) peptide, with no acetylation, from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the VP3 capsid protein without N-term acetylation identified during intact mass analysis (Table S8) Figure S13 MS/MS spectra of the VEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPD (V132-D185) peptide, with phosphorylation around S149 (exact location could not be determined), from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the V132-VP1 fragment identified during intact mass analysis (Table S8).The relatively poor spectrum is a result of the phosphorylation present which impacts digestion efficiency of the pepsin, resulting in the large peptide identified

1104.9720
Figure S14 MS/MS spectra of the GAPMADNNEGADGVGSSSGNWHCDSTWLGDRVIT (G209-T242) peptide from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the G209-VP3 fragment identified during intact mass analysis (Table S8

G209-VP3 G209-T242
Figure S15 MS/MS spectra of the AADGYLPDWLEDNLSEGIRE (A2-E21) peptide, with acetylation at A2, from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP1 capsid protein identified during intact mass analysis (Table S9

(Ac)VP1 A2(Ac)-E21
Figure S16 MS/MS spectra of the APGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTM (A139-M203) peptide, from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the VP2 capsid protein identified during intact mass analysis (Table S9)

VP2 A139-M203
Figure S17 MS/MS spectra of the ASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVIT (A204-T241) peptide, with acetylation at A204, from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3 capsid protein identified during intact mass analysis (Table S9 (Ac)VP3 A204(Ac)-T241

Relative Abundance
Figure S18 MS/MS spectra of the MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRT (M203-T246) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the M203-VP3 capsid protein identified during intact mass analysis (Table S9).This is a VP3 protein that didn't undergo expected N-term methionine cleavage and subsequent N-term acetylation

M203-VP3 M203-T246
Figure S19 MS/MS spectra of the RAVFQAKKRLLEPLGL (R116-L131) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the R116-VP1 fragment identified during intact mass analysis (Table S9)

Relative Abundance
Figure S20 MS/MS spectra of the LVEEAAKTAPGKKRPVE (L131-E147) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the L131-VP1 fragment identified during intact mass analysis (Table S9)

Relative Abundance
Figure S21 MS/MS spectra of the FGQTGDTESVPDPQPIGEPPAAPSGVGSL (F173-L201) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the F173-VP2 fragment identified during intact mass analysis (Table S9

Relative Abundance
Figure S23 MS/MS spectra of the ALNGRNSLMNPGPAMASHKEGEDRFFPLS (A510-S538) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.
Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-S538 fragment identified during intact mass analysis (Table S9)  S24 MS/MS spectra of the FAWPGASSWALNGRNSLM (F501-M518) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-M518 fragment identified during intact mass analysis (Table S9 (Ac)VP3-M518 F501-M518

Relative Abundance
Figure S25 MS/MS spectra of the AVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALN (A472-N512) peptide from peptide mapping of full AAV9 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3-N512 fragment identified during intact mass analysis (Table S9) (Ac)VP3-N512 A472-N512

Table S1 :
BPF 5.1 software parameter settings for intact mass analysis

Table S2 :
BPF 5.1 software parameter settings for peptide mapping analysis

Table S4 :
Identified AAV8 viral capsid proteins (VPs) under investigated sample analysis conditions

Table S5 :
Empty AAV8 capsid dilution series for ZipChip limit of detection (LoD) evaluation

Table S1 :
Unknown components detected in the empty and full capsids of AAV6, AAV8, and AAV9

Table S7 :
Select peptides found in both empty and full AAV6 peptide mapping analysis to confirm presence of VPs and fragments detected during intact mass analysis.Tandem MS (MS/MS) spectra of the highlighted peptides is shown in FiguresS2-S7as illustrative examples of the quality of the peptides identified for each VP or fragment.Peptide information displayed is from the analysis of the full AAV6 capsids

Table S8 :
Select peptides found in both empty and full AAV8 peptide mapping analysis to confirm presence of VPs and fragments detected during intact mass analysis.MS/MS spectra of the highlighted peptides is shown in FiguresS8-S14as illustrative examples of the quality of the peptides identified for each VP or fragment.Peptide information displayed is from the analysis of the full AAV8 capsids

Table S9 :
Select peptides found in both empty and full AAV9 peptide mapping analysis to confirm presence of VPs and fragments detected during intact mass analysis.MS/MS spectra of the highlighted peptides is shown in FiguresS15-S26as illustrative examples of the quality of the peptides identified for each VP or fragment.Peptide information displayed is from the analysis of the full AAV9 capsids

Table S10 :
Acetylation and phosphorylation PTMs identified during peptide mapping of AAV6 empty and full capsids with a relative abundance ≥ 1%.~ indicates that the exact location of the modification could not be determined by BPF 5.1, but the modification is near or on the listed residue

Table S11 :
Acetylation and phosphorylation PTMs identified during peptide mapping of AAV8 empty and full capsids with a relative abundance ≥ 1%.~ indicates that the exact location of the modification could not be determined by BPF 5.1, but the modification is near or on the listed residue

Table S12 :
Acetylation and phosphorylation PTMs identified during peptide mapping of AAV9 empty and full capsids with a relative abundance ≥ 1%.~ indicates that the exact location of the modification could not be determined by BPF 5.1, but the modification is near or on the listed residue Figure S1 Plot of total ion electropherogram area (blue line on top) and extracted apex MS signal intensity (orange line on bottom) versus Vps injected during LoD study

Apex MS Spectra Intensity (Spectral units) Total Ion Electropherogram Area (counts*min) Amount of AAV Injected (viral particles) Area and Intensity vs AAV Viral Particle Quantities
Figure S2Tandem MS (MS/MS) spectra of the AADGYLPDWLEDNLSEGIRE (A2-E21) peptide, with acetylation at A2, from peptide mapping of full AAV6 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP1 capsid protein identified during intact mass analysis (TableS7) ) ) Figure S12MS/MS spectra of the ADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRT (A213-T247) peptide, with acetylation at A213, from peptide mapping of full AAV8 capsids, as identified using BPF 5.1.Peptide is an example of the detected peptides that indicate the presence of the (Ac)VP3 Variant capsid protein identified during intact mass analysis (TableS8)