An Internal Standard for Assessing Phosphopeptide Recovery from Metal Ion/Oxide Enrichment Strategies

Abstract

Phosphorylation-mediated signaling pathways have major implications in cellular regulation and disease. However, proteins with roles in these pathways are frequently less abundant and phosphorylation is often sub-stoichiometric. As such, the efficient enrichment, and subsequent recovery of phosphorylated peptides, is vital. Mass spectrometry-based proteomics is a well-established approach for quantifying thousands of phosphorylation events in a single experiment. We designed a peptide internal standard-based assay directed toward sample preparation strategies for mass spectrometry analysis to understand better phosphopeptide recovery from enrichment strategies. We coupled mass-differential tandem mass tag (mTMT) reagents (specifically, TMTzero and TMTsuper-heavy), nine mass spectrometry-amenable phosphopeptides (phos9), and peak area measurements from extracted ion chromatograms to determine phosphopeptide recovery. We showcase this mTMT/phos9 recovery assay by evaluating three phosphopeptide enrichment workflows. Our assay provides data on the recovery of phosphopeptides, which complement other metrics, namely the number of identified phosphopeptides and enrichment specificity. Our mTMT/phos9 assay is applicable to any enrichment protocol in a typical experimental workflow irrespective of sample origin or labeling strategy. Graphical Abstract ᅟ.

Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3

“…Recently, simple and efficient centrifugation-based phosphopeptide enrichment has been introduced commercially as the Pierce Fe-NTA Phosphopeptide Enrichment Kit (Figure 1G), which has been shown to have a very high recovery rate (22). These convenient IMAC spin columns can process 50 μg to 5 mg of peptide and can yield up to 150 μg of phosphopeptides.…”

A guanidyl-functionalized TiO2 nanoparticle-anchored graphene nanohybrid for enhanced capture of phosphopeptides

“…TiO 2 -based metal oxide affinity chromatography (MOAC) nanomaterials are regarded as one of the most promising materials for phosphopeptide enrichment due to the specic chemisorption toward phosphate groups. [1][2][3][4] However, the serious non-specic adsorption of acidic peptides greatly reduces the specicity for enrichment of phosphopeptides and the limited surface area also results in a limited chemisorption performance of phosphopeptides. Although there exists some drawbacks for the TiO 2 -based MOAC nanomaterials in the enrichment of phosphopeptides, the performance of TiO 2based materials toward phosphopeptide enrichment is still better than that of conventional enrichment materials.…”

Advances in quantitative high‐throughput phosphoproteomics with sample multiplexing

“…and Fe-NTA [37]. Modern IMAC (Fe-NTA) spin columns provide a balance between recovery, enrichment, throughput and efficiency [38]. These simple and efficient centrifugation-based phosphopeptide enrichment columns have been introduced commercially as well (e.g., Pierce Fe-NTA Phosphopeptide Enrichment Kit).…”

“…These simple and efficient centrifugation-based phosphopeptide enrichment columns have been introduced commercially as well (e.g., Pierce Fe-NTA Phosphopeptide Enrichment Kit). The commercial options have also been shown to have a high recovery rate for phosphopeptides [39]. Furthermore, they enable processing of 50 μg to 5 mg of peptide and can yield up to 150 μg of phosphopeptides per column.…”

“…Such analysis permits the survey of several thousand phosphorylation sites with nominal cost and with minimal effort [38]. Yet, the adoption of the "mini-phos" enrichment is still hampered by a lack of depth due to the relatively low amount of analyte compared to larger-scale studies that enrich from several milligrams of protein lysate [46].…”