Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics
A recent study demonstrated a substantial increase in the peptide signal and corresponding proteome coverage when employing 0.5% acetic acid (AA) as the ion pairing modifier in place of the 0.1% formic acid traditionally used in shotgun proteomics. Given the strictly limited material and counterintu...
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2025
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| _version_ | 1849927644767846400 |
|---|---|
| author | Colten D. Eberhard (17729446) |
| author2 | Cameron Braswell (22674649) Benjamin C. Orsburn (9306361) |
| author2_role | author author |
| author_facet | Colten D. Eberhard (17729446) Cameron Braswell (22674649) Benjamin C. Orsburn (9306361) |
| author_role | author |
| dc.creator.none.fl_str_mv | Colten D. Eberhard (17729446) Cameron Braswell (22674649) Benjamin C. Orsburn (9306361) |
| dc.date.none.fl_str_mv | 2025-11-24T12:49:00Z |
| dc.identifier.none.fl_str_mv | 10.1021/acs.jproteome.5c00930.s001 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/dataset/Alternative_Ion-Pairing_Modifiers_Should_Be_Investigated_in_Low-Input_and_Single-Cell_Proteomics/30694666 |
| dc.rights.none.fl_str_mv | CC BY-NC 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biophysics Biochemistry Medicine Biological Sciences not elsewhere classified Chemical Sciences not elsewhere classified subnanogram proteome dilutions strictly limited material reduce ramp times recent study demonstrated increased proteome coverage corresponding proteome coverage 30 min analysis ion pairing modifiers ion pairing modifier pairing modifiers alternative ion alternative modifier vendor raw shotgun proteomics scans across processed data peptide groups lower concentrations emerging field digest standards counterintuitive observations cell proteomics approximately 13 additives warrant acetic acid 7 × |
| dc.title.none.fl_str_mv | Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics |
| dc.type.none.fl_str_mv | Dataset info:eu-repo/semantics/publishedVersion dataset |
| description | A recent study demonstrated a substantial increase in the peptide signal and corresponding proteome coverage when employing 0.5% acetic acid (AA) as the ion pairing modifier in place of the 0.1% formic acid traditionally used in shotgun proteomics. Given the strictly limited material and counterintuitive observations by others in the emerging field of single-cell proteomics, we chose to investigate this alternative modifier in the analysis of subnanogram proteome dilutions. When digest standards as low as 20 pg total load on the column were evaluated, AA led to increased proteome coverage at every peptide load assessed. Relative improvements were more apparent at lower concentrations, with a 20 pg peptide digest demonstrating a striking 1.8-fold increase to over 2000 protein groups identified in a 30 min analysis. Furthermore, we find that this increase in signal can be leveraged to reduce ramp times, leading to 1.7× more scans across each peak and improvements in quantification, as measured by %CVs. These results can be reproduced on multiple trapped ion mobility instruments. When evaluating single cancer cells, approximately 13% more peptide groups were identified on average when employing AA in the place of FA. These results suggest that ion pairing modifiers and other additives warrant re-evaluation in the context of low-input and single-cell proteomics. All vendor raw and processed data are available through ProteomeXchange as PXD046002 and PXD051590. |
| eu_rights_str_mv | openAccess |
| id | Manara_7febb403c6d07d1a4bd4df57a8f9d379 |
| identifier_str_mv | 10.1021/acs.jproteome.5c00930.s001 |
| network_acronym_str | Manara |
| network_name_str | ManaraRepo |
| oai_identifier_str | oai:figshare.com:article/30694666 |
| publishDate | 2025 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY-NC 4.0 |
| spelling | Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell ProteomicsColten D. Eberhard (17729446)Cameron Braswell (22674649)Benjamin C. Orsburn (9306361)BiophysicsBiochemistryMedicineBiological Sciences not elsewhere classifiedChemical Sciences not elsewhere classifiedsubnanogram proteome dilutionsstrictly limited materialreduce ramp timesrecent study demonstratedincreased proteome coveragecorresponding proteome coverage30 min analysision pairing modifiersion pairing modifierpairing modifiersalternative ionalternative modifiervendor rawshotgun proteomicsscans acrossprocessed datapeptide groupslower concentrationsemerging fielddigest standardscounterintuitive observationscell proteomicsapproximately 13additives warrantacetic acid7 ×A recent study demonstrated a substantial increase in the peptide signal and corresponding proteome coverage when employing 0.5% acetic acid (AA) as the ion pairing modifier in place of the 0.1% formic acid traditionally used in shotgun proteomics. Given the strictly limited material and counterintuitive observations by others in the emerging field of single-cell proteomics, we chose to investigate this alternative modifier in the analysis of subnanogram proteome dilutions. When digest standards as low as 20 pg total load on the column were evaluated, AA led to increased proteome coverage at every peptide load assessed. Relative improvements were more apparent at lower concentrations, with a 20 pg peptide digest demonstrating a striking 1.8-fold increase to over 2000 protein groups identified in a 30 min analysis. Furthermore, we find that this increase in signal can be leveraged to reduce ramp times, leading to 1.7× more scans across each peak and improvements in quantification, as measured by %CVs. These results can be reproduced on multiple trapped ion mobility instruments. When evaluating single cancer cells, approximately 13% more peptide groups were identified on average when employing AA in the place of FA. These results suggest that ion pairing modifiers and other additives warrant re-evaluation in the context of low-input and single-cell proteomics. All vendor raw and processed data are available through ProteomeXchange as PXD046002 and PXD051590.2025-11-24T12:49:00ZDatasetinfo:eu-repo/semantics/publishedVersiondataset10.1021/acs.jproteome.5c00930.s001https://figshare.com/articles/dataset/Alternative_Ion-Pairing_Modifiers_Should_Be_Investigated_in_Low-Input_and_Single-Cell_Proteomics/30694666CC BY-NC 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/306946662025-11-24T12:49:00Z |
| spellingShingle | Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics Colten D. Eberhard (17729446) Biophysics Biochemistry Medicine Biological Sciences not elsewhere classified Chemical Sciences not elsewhere classified subnanogram proteome dilutions strictly limited material reduce ramp times recent study demonstrated increased proteome coverage corresponding proteome coverage 30 min analysis ion pairing modifiers ion pairing modifier pairing modifiers alternative ion alternative modifier vendor raw shotgun proteomics scans across processed data peptide groups lower concentrations emerging field digest standards counterintuitive observations cell proteomics approximately 13 additives warrant acetic acid 7 × |
| status_str | publishedVersion |
| title | Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics |
| title_full | Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics |
| title_fullStr | Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics |
| title_full_unstemmed | Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics |
| title_short | Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics |
| title_sort | Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics |
| topic | Biophysics Biochemistry Medicine Biological Sciences not elsewhere classified Chemical Sciences not elsewhere classified subnanogram proteome dilutions strictly limited material reduce ramp times recent study demonstrated increased proteome coverage corresponding proteome coverage 30 min analysis ion pairing modifiers ion pairing modifier pairing modifiers alternative ion alternative modifier vendor raw shotgun proteomics scans across processed data peptide groups lower concentrations emerging field digest standards counterintuitive observations cell proteomics approximately 13 additives warrant acetic acid 7 × |