AI Article Synopsis
- The study investigates automated phosphopeptide enrichment using two methods: Immobilized Metal Affinity Chromatography (IMAC) with POROS-Fe³⁺ and Metal Oxide Affinity Chromatography (MOAC) with TiO₂, focusing on peptide samples from casein-albumin and mouse liver.
- Selectivity of both methods was found to be pH-dependent, with notable increases when using 0.1 M acetic acid or trifluoroacetic acid, especially in complex liver extracts.
- However, while the TiO₂ columns maintained stability and reliability, the POROS-Fe³⁺ columns experienced a decrease in phosphopeptide identification due to strong Fe³⁺ leaching and
Article Abstract
Automated phosphopeptide enrichment prior to MS analysis by means of Immobilized Metal Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC) has been probed with packed columns. We compared POROS-Fe³⁺ and TiO₂ (respectively IMAC and MOAC media), using a simple mixture of peptides from casein-albumin and a complex mixture of peptides isolated from mouse liver. With theses samples, selectivity of POROS-Fe³⁺ and TiO₂ were pH dependant. In the case of liver extract, selectivity increased from 12-18% to 58-60% when loading buffer contained 0.1 M acetic acid or 0.1 M trifluoroacetic acid, respectively. However, with POROS-Fe³⁺ column, the number of identifications decreased from 356 phosphopeptides with 0.1 M acetic acid to 119 phosphopeptides with 0.1 M TFA. This decrease of binding capacity of POROS-Fe³⁺ was associated with strong Fe³⁺ leaching. Furthermore, repetitive use of IMAC-Fe³⁺ with the 0.5 M NH₄OH solution required for phosphopeptide elution induced Fe₂O₃ accumulation in the column. By comparison, MOAC columns packed with TiO₂ support do not present any problem of stability in the same conditions and provide a reliable solution for packed column phosphopeptide enrichment.
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| http://dx.doi.org/10.1016/j.jchromb.2012.02.028 | DOI Listing |
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