SIMAC (Sequential Elution from IMAC), a Phosphoproteomics Strategy for the Rapid Separation of Monophosphorylated from Multiply Phosphorylated Peptides

Abstract

The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 g of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SI-

Analytical strategies for phosphoproteomics

“…A general assumption has been that TiO 2 chromatography has a preference for mono-phosphorylated peptides, and the level of multiply phosphorylated peptides identified by TiO 2 chromatography is consequently rather low. We believe that TiO 2 does, in fact, bind multiply phosphorylated peptides as well as mono-phosphorylated peptides, but that they are difficult to elute due to their extremely high binding affinity [74]. On the other hand, IMAC, has been argued to be less efficient for large-scale phosphoproteomic studies, and to have a preference for multiply phosphorylated peptides [48,73,74].…”

“…In this offline strategy, peptide loading is performed at highly acidic loading conditions using various substituted organic acids including 2,5-dihydroxybenzoic acid (DHB), phthalic acid or glycolic acid to efficiently eliminate binding of acidic peptides to the TiO 2 resin [73][74][75][76]. In addition, by increasing pH of the elution buffer from 9.0 to 11.3 (using ammonia solution), phosphopeptides are more efficiently eluted from the TiO 2 resin, resulting in increased sensitivity [75].…”

“…Due to the low intensities of multiply phosphorylated peptide ions, they are rarely selected for fragmentation during the analysis of complex samples, which is why mono-phosphorylated peptides are predominantly identified in large-scale experiments, particularly when TiO 2 is used for phosphopeptide enrichment. In 2007, our group presented SIMAC (Sequential elution from IMAC), a new method, which combines the strengths of IMAC with the strengths of TiO 2 [74]. SIMAC is a simple and straight forward strategy, in which mono-and multiply phosphorylated peptides are efficiently enriched from highly complex samples into separate pools, which can be analyzed using different MS/MS settings targeting either mono-phosphorylated or multiply phosphorylated peptide ions.…”

Mass spectrometric analysis of mono- and multi-phosphopeptides by selective binding with NiZnFe2O4 magnetic nanoparticles

“…The performance of NiZnFe 2 O 4 has also been compared with other approaches including TiO 2 27-30 , Fe 3 þ -IMAC 31 and HAP (hydroxyapatite) 32,33 (Supplementary Note 2; Supplementary Table S2). These materials can bind with both mono-and multi-phosphorylated peptides without selectivity ( Supplementary Fig.…”

Mesoporous TiO2-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

“…In order to enrich the phosphopeptides most of the new methods make use of the high affinity of the phosphoryl group to metals and metal oxides (e.g., on immobilized metal affinity chromatography (IMAC) [2][3][4][5] , metal oxide affinity chromatography (MOAC) 1,[6][7][8][9][10][11][12][13][14] , sequential immobilized metal affinity chromatography (SIMAC) 15 and direct on-target enrichment [16][17][18][19][20][21][22][23] ). Mass spectrometry (MS) is then commonly used to analyze the peptides.…”