Development of automated proteomic workflows utilizing silicon-based coupling agents Article Swipe
Connor Frey
,
Maor Arad
,
Kenneth Ku
,
Rhien Hare
,
Ronald Balagtas
,
Yuming Shi
,
Kyung‐Mee Moon
,
Leonard J. Foster
,
Golfam Ghafourifar
·
YOU?
·
· 2024
· Open Access
·
· DOI: https://doi.org/10.1016/j.jprot.2024.105215
YOU?
·
· 2024
· Open Access
·
· DOI: https://doi.org/10.1016/j.jprot.2024.105215
Automated methods for enzyme immobilization via 4-triethoxysilylbutyraldehyde (TESB) derived silicone-based coupling agents were developed. TESB and its oxidized derivative, 4-triethoxysilylbutanoic acid (TESBA), were determined to be the most effective. The resulting immobilized enzyme particles (IEPs) displayed robustness, rapid digestion, and immobilization efficiency of 51 ± 8%. Furthermore, we automated the IEP procedure, allowing for multiple enzymes, and/or coupling agents to be fabricated at once, in a fraction of the time via an Agilent Bravo. The automated trypsin TESB and TESBA IEPs were shown to rival a classical in-gel digestion method. Moreover, pepsin IEPs favored cleavage at leucine (>50%) over aromatic and methionine residues. The IEP method was then adapted for an in-situ immobilized enzyme microreactor (IMER) fabrication. We determined that TESBA could functionalize the silica capillary's inner wall while simultaneously acting as an enzyme coupler. The IMER digestion of bovine serum albumin (BSA), mirroring IEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as little as 15 min. Overall, our findings underscore the potential of both IEP and IMER methods, paving the way for automated analysis and a reduction in enzyme waste through reuse, thereby contributing to a more cost-effective and timely study of the proteome. SIGNIFICANCE: This research introduces 4-triethoxysilylbutyraldehyde (TESB) and its derivatives as silicon-based enzyme coupling agents and an automated liquid handling method for bottom-up proteomics (BUP) while streamlining sample preparation for high-throughput processing. Additionally, immobilized enzyme particle (IEP) fabrication and digestion within the 96-well plate allows for flexibility in protocol where different enzyme-coupler combinations can be employed simultaneously. By enabling the digestion of entire microplates and reducing manual labor, the proposed method enhances reproducibility and offers a more efficient alternative to classical in-gel techniques. Furthermore, pepsin IEPs were noted to favor cleavage at leucine residues which represents an interesting finding when compared to the literature that warrants further study. The capability of immobilized enzyme microreactors (IMER) for rapid digestion (in as little as 15 min) demonstrated the system's efficiency and potential for rapid proteomic analysis. This advancement in BUP not only improves efficiency, but also opens avenues for a fully automated, mass spectrometry-integrated proteomics workflow, promising to expedite research and discoveries in complex biological studies.
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Metadata
- Type
- article
- Language
- en
- Landing Page
- https://doi.org/10.1016/j.jprot.2024.105215
- OA Status
- hybrid
- Cited By
- 2
- References
- 64
- Related Works
- 10
- OpenAlex ID
- https://openalex.org/W4399361084
All OpenAlex metadata
Raw OpenAlex JSON
- OpenAlex ID
-
https://openalex.org/W4399361084Canonical identifier for this work in OpenAlex
- DOI
-
https://doi.org/10.1016/j.jprot.2024.105215Digital Object Identifier
- Title
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Development of automated proteomic workflows utilizing silicon-based coupling agentsWork title
- Type
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articleOpenAlex work type
- Language
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enPrimary language
- Publication year
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2024Year of publication
- Publication date
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2024-06-05Full publication date if available
- Authors
-
Connor Frey, Maor Arad, Kenneth Ku, Rhien Hare, Ronald Balagtas, Yuming Shi, Kyung‐Mee Moon, Leonard J. Foster, Golfam GhafourifarList of authors in order
- Landing page
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https://doi.org/10.1016/j.jprot.2024.105215Publisher landing page
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YesWhether a free full text is available
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hybridOpen access status per OpenAlex
- OA URL
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https://doi.org/10.1016/j.jprot.2024.105215Direct OA link when available
- Concepts
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Chemistry, Chromatography, Immobilized enzyme, Trypsin, Bovine serum albumin, Proteome, Combinatorial chemistry, Enzyme, BiochemistryTop concepts (fields/topics) attached by OpenAlex
- Cited by
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2Total citation count in OpenAlex
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2024: 2Per-year citation counts (last 5 years)
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64Number of works referenced by this work
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10Other works algorithmically related by OpenAlex
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| abstract_inverted_index.(>50%) | 97 |
| abstract_inverted_index.(BSA), | 142 |
| abstract_inverted_index.(IEPs) | 34 |
| abstract_inverted_index.(IMER) | 115, 313 |
| abstract_inverted_index.(TESB) | 7, 205 |
| abstract_inverted_index.33-40% | 149 |
| abstract_inverted_index.Bravo. | 73 |
| abstract_inverted_index.acting | 130 |
| abstract_inverted_index.agents | 11, 58, 213 |
| abstract_inverted_index.allows | 243 |
| abstract_inverted_index.and/or | 56 |
| abstract_inverted_index.bovine | 139 |
| abstract_inverted_index.entire | 261 |
| abstract_inverted_index.enzyme | 3, 32, 113, 133, 184, 211, 233, 311 |
| abstract_inverted_index.in-gel | 87, 280 |
| abstract_inverted_index.labor, | 266 |
| abstract_inverted_index.liquid | 217 |
| abstract_inverted_index.little | 158, 319 |
| abstract_inverted_index.manual | 265 |
| abstract_inverted_index.method | 105, 219, 269 |
| abstract_inverted_index.offers | 273 |
| abstract_inverted_index.paving | 174 |
| abstract_inverted_index.pepsin | 91, 283 |
| abstract_inverted_index.reuse, | 187 |
| abstract_inverted_index.sample | 226 |
| abstract_inverted_index.silica | 124 |
| abstract_inverted_index.study. | 306 |
| abstract_inverted_index.timely | 195 |
| abstract_inverted_index.within | 239 |
| abstract_inverted_index.96-well | 241 |
| abstract_inverted_index.Agilent | 72 |
| abstract_inverted_index.adapted | 108 |
| abstract_inverted_index.albumin | 141 |
| abstract_inverted_index.avenues | 344 |
| abstract_inverted_index.complex | 360 |
| abstract_inverted_index.derived | 8 |
| abstract_inverted_index.favored | 93 |
| abstract_inverted_index.finding | 297 |
| abstract_inverted_index.further | 305 |
| abstract_inverted_index.in-situ | 111 |
| abstract_inverted_index.leucine | 96, 291 |
| abstract_inverted_index.method. | 89 |
| abstract_inverted_index.methods | 1 |
| abstract_inverted_index.primary | 150 |
| abstract_inverted_index.thereby | 188 |
| abstract_inverted_index.through | 186 |
| abstract_inverted_index.trypsin | 76 |
| abstract_inverted_index.yielded | 147 |
| abstract_inverted_index.(TESBA), | 21 |
| abstract_inverted_index.LC-MS/MS | 154 |
| abstract_inverted_index.Overall, | 162 |
| abstract_inverted_index.allowing | 52 |
| abstract_inverted_index.analysis | 155, 179 |
| abstract_inverted_index.aromatic | 99 |
| abstract_inverted_index.cleavage | 94, 289 |
| abstract_inverted_index.compared | 299 |
| abstract_inverted_index.coupler. | 134 |
| abstract_inverted_index.coupling | 10, 57, 212 |
| abstract_inverted_index.coverage | 152 |
| abstract_inverted_index.employed | 254 |
| abstract_inverted_index.enabling | 257 |
| abstract_inverted_index.enhances | 270 |
| abstract_inverted_index.enzymes, | 55 |
| abstract_inverted_index.expedite | 355 |
| abstract_inverted_index.findings | 164 |
| abstract_inverted_index.fraction | 66 |
| abstract_inverted_index.handling | 218 |
| abstract_inverted_index.improves | 339 |
| abstract_inverted_index.methods, | 173 |
| abstract_inverted_index.multiple | 54 |
| abstract_inverted_index.oxidized | 17 |
| abstract_inverted_index.particle | 234 |
| abstract_inverted_index.proposed | 268 |
| abstract_inverted_index.protocol | 247 |
| abstract_inverted_index.reducing | 264 |
| abstract_inverted_index.research | 202, 356 |
| abstract_inverted_index.residues | 292 |
| abstract_inverted_index.sequence | 151 |
| abstract_inverted_index.studies. | 362 |
| abstract_inverted_index.system's | 325 |
| abstract_inverted_index.warrants | 304 |
| abstract_inverted_index.Automated | 0 |
| abstract_inverted_index.Moreover, | 90 |
| abstract_inverted_index.analysis. | 332 |
| abstract_inverted_index.automated | 48, 75, 178, 216 |
| abstract_inverted_index.bottom-up | 221 |
| abstract_inverted_index.classical | 86, 279 |
| abstract_inverted_index.different | 249 |
| abstract_inverted_index.digestion | 88, 137, 145, 238, 259, 316 |
| abstract_inverted_index.displayed | 35 |
| abstract_inverted_index.efficient | 276 |
| abstract_inverted_index.mirroring | 143 |
| abstract_inverted_index.particles | 33 |
| abstract_inverted_index.potential | 167, 328 |
| abstract_inverted_index.promising | 353 |
| abstract_inverted_index.proteome. | 199 |
| abstract_inverted_index.proteomic | 331 |
| abstract_inverted_index.reduction | 182 |
| abstract_inverted_index.residues. | 102 |
| abstract_inverted_index.resulting | 30 |
| abstract_inverted_index.workflow, | 352 |
| abstract_inverted_index.automated, | 348 |
| abstract_inverted_index.biological | 361 |
| abstract_inverted_index.capability | 308 |
| abstract_inverted_index.determined | 23, 118 |
| abstract_inverted_index.developed. | 13 |
| abstract_inverted_index.digestion, | 38 |
| abstract_inverted_index.effective. | 28 |
| abstract_inverted_index.efficiency | 41, 326 |
| abstract_inverted_index.fabricated | 61 |
| abstract_inverted_index.introduces | 203 |
| abstract_inverted_index.literature | 302 |
| abstract_inverted_index.methionine | 101 |
| abstract_inverted_index.procedure, | 51 |
| abstract_inverted_index.proteomics | 222, 351 |
| abstract_inverted_index.represents | 294 |
| abstract_inverted_index.underscore | 165 |
| abstract_inverted_index.advancement | 334 |
| abstract_inverted_index.alternative | 277 |
| abstract_inverted_index.capillary's | 125 |
| abstract_inverted_index.conditions, | 146 |
| abstract_inverted_index.derivative, | 18 |
| abstract_inverted_index.derivatives | 208 |
| abstract_inverted_index.discoveries | 358 |
| abstract_inverted_index.efficiency, | 340 |
| abstract_inverted_index.fabrication | 236 |
| abstract_inverted_index.flexibility | 245 |
| abstract_inverted_index.immobilized | 31, 112, 232, 310 |
| abstract_inverted_index.interesting | 296 |
| abstract_inverted_index.microplates | 262 |
| abstract_inverted_index.preparation | 227 |
| abstract_inverted_index.processing. | 230 |
| abstract_inverted_index.robustness, | 36 |
| abstract_inverted_index.techniques. | 281 |
| abstract_inverted_index.Furthermore, | 46, 282 |
| abstract_inverted_index.combinations | 251 |
| abstract_inverted_index.contributing | 189 |
| abstract_inverted_index.demonstrated | 323 |
| abstract_inverted_index.fabrication. | 116 |
| abstract_inverted_index.microreactor | 114 |
| abstract_inverted_index.streamlining | 225 |
| abstract_inverted_index.Additionally, | 231 |
| abstract_inverted_index.SIGNIFICANCE: | 200 |
| abstract_inverted_index.functionalize | 122 |
| abstract_inverted_index.microreactors | 312 |
| abstract_inverted_index.silicon-based | 210 |
| abstract_inverted_index.cost-effective | 193 |
| abstract_inverted_index.enzyme-coupler | 250 |
| abstract_inverted_index.immobilization | 4, 40 |
| abstract_inverted_index.silicone-based | 9 |
| abstract_inverted_index.simultaneously | 129 |
| abstract_inverted_index.high-throughput | 229 |
| abstract_inverted_index.reproducibility | 271 |
| abstract_inverted_index.simultaneously. | 255 |
| abstract_inverted_index.spectrometry-integrated | 350 |
| abstract_inverted_index.4-triethoxysilylbutanoic | 19 |
| abstract_inverted_index.4-triethoxysilylbutyraldehyde | 6, 204 |
| cited_by_percentile_year.max | 96 |
| cited_by_percentile_year.min | 94 |
| corresponding_author_ids | https://openalex.org/A5058742097 |
| countries_distinct_count | 2 |
| institutions_distinct_count | 9 |
| corresponding_institution_ids | https://openalex.org/I170606269 |
| citation_normalized_percentile.value | 0.64292393 |
| citation_normalized_percentile.is_in_top_1_percent | False |
| citation_normalized_percentile.is_in_top_10_percent | False |