An immobilized titanium (IV) ion affinity chromatography adsorbent for solid phase extraction of phosphopeptides for phosphoproteome analysis

Highlights

• A new Ti⁴⁺-IMAC adsorbent was synthesized and applied for phosphopeptide enrichment.

• It exhibited excellent specificity of over 99% for the enrichment of phosphopeptides.

• The spin tip was fitted for the analysis of minute amount of complex samples.

Abstract

In this study, we developed a centrifugation assisted solid phase extraction (SPE) method for the selective enrichment of phosphopeptides using a new Ti⁴⁺-IMAC material synthesized in-house. This new material has the feature of big size and large specific surface area which makes it more suitable to enrich phosphopeptides in a SPE way. The spin tips loaded with the Ti⁴⁺-IMAC material were applied to enrich phosphopeptides from the complex protein digests. It was found that phosphopeptides can be specifically enriched from tryptic digest of bovine serum albumin and β-casein at a molar ratio up to 1000:1. And about 4700 unique phosphorylated peptides can be identified with the specificity as high as 99% from the tryptic digest of HeLa cell proteins. This tip was demonstrated to have good column-to-column reproducibility. Furthermore, it is fitted to analyze minute amount of sample. Compared with the conventional solution method, the SPE method facilitated the rapid and complete separation of the material with solution, which making it a time-saving and convenient method for phosphopeptide enrichment. Compared with the commercial TiO₂ material, this new materials yielded much more phosphopeptide identifications and much higher enrichment specificity.

Introduction

As one of the most important post-translational modifications (PTMs), protein phosphorylation plays key roles in the dynamic regulation of many cellular processes, such as cell division, cell growth, signal transduction, apoptosis, and so forth [1], [2], [3]. Aberrant phosphorylation caused by oncogenic kinase signaling has been found in many human diseases including malignant tumors [4], [5]. Therefore, to fundamentally understand the signaling networks and biological processes, a detailed analysis of the involved phosphorylated proteins is essential. Nowadays, high-throughput protein identification is predominantly accomplished by mass spectrometry (MS) based techniques [6], [7]. However, the low abundance, low ionization efficiency of phosphopeptides, and the serious interference of unphosphorylated peptides greatly limit the comprehensive characterization of protein phosphorylation at proteome level. Hence, specifically isolation of phosphopeptides from complex peptide mixtures is the critical step for phosphoproteomics analysis.

Various metal oxides were used for the enrichment of phosphopeptides. Among them, TiO₂ is the most frequently used one due to its good performance [3], [8]. However, this method usually requires 2,5-dihydroxybenzoic acid (DHB), phthalic acid or other acidic reagents as modifiers to enhance the purification efficiency [9], [10], [11]. Immobilized metal ion affinity chromatography (IMAC), mainly Fe³⁺-IMAC with either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) as chelating group, was often used to enrich phosphopeptides [12], [13], [14], [15], [16], [17]. However, this conventional Fe³⁺-IMAC suffers from the poor specificity and is often bias to multiply phosphorylated peptides [18]. Eight years ago, a new generation of IMAC with super specificity, i.e. immobilized titanium (IV) ion affinity chromatography (Ti⁴⁺-IMAC), was developed by our lab [19], [20]. Instead of using IDA or NTA, phosphate group is used as the chelating group to immobilize Ti⁴⁺ because of its strong interaction with Ti⁴⁺. The phosphopeptides are specifically captured by Ti⁴⁺-IMAC also due to the strong chelating interaction between the phosphate groups on the peptides and the immobilized Ti⁴⁺. Ti⁴⁺-IMAC has become a popular method for phosphopeptide enrichment because of its superior performance [10], [21], [22], [23]. In 2009, we developed monodisperse Ti⁴⁺-IMAC microspheres for specific enrichment of phosphopeptides. The protocol for the synthesis and application of this material was published in Nature Protocols in 2013 [24]. The size of this monodisperse microspheres was about 12 μm, so that the resulting Ti⁴⁺-IMAC materials were best fitted to process the phosphopeptide enrichment in solution by centrifuge. This enrichment mode, requiring incubation and centrifugation procedures, is time-consuming and not fitted to analyze minute amount of samples. We attempted to pack these resins into tip for enrichment of phosphopeptide in solid-phase extraction (SPE) mode. However, the backpressure for the resulting tip is very high and the sample as well as solvents flow very slow through the tip even the tip is placed in a tube via centrifuge. Instead, we recently developed a monolithic polypropylene pipette tip grafted with porous Ti (IV) monolithic materials for the enrichment of phosphopeptides [25]. Due to the low back pressure, the phosphopeptide enrichment by this tip can be performed in a SPE mode, where the separation of solution with material could be easily achieved. However, the maximum capacity of the Ti (IV) monolithic tip is 25 μg, indicating that this tip is only applicable for the analysis of minute amount of biological samples.

In this study, we synthesized a new Ti⁴⁺-IMAC adsorbent and applied it as packing material to prepare spin tips for phosphopeptide enrichment in a centrifugation assisted SPE way. Compared with the conventional Ti⁴⁺-IMAC adsorbent, this newly prepared material has bigger size and larger specific surface area, making it an ideal candidate to act as SPE packing material. The performance of this new Ti⁴⁺-IMAC material was evaluated by analyzing different biological samples and found to be as excellent as the small size Ti⁴⁺-IMAC material. When the material was applied for the analysis of human HeLa cell phosphoproteome, over 4700 phosphorylation peptides were identified with an enrichment specificity over 99%. Similar to the Ti (IV) monolithic tip [25], this Ti⁴⁺-IMAC packed tip can also be used for the analysis of minute amount of samples. About 900 unique phosphopeptides were identified from 5 μg HeLa cell digest, with the enrichment specificity of 85%. Compared with the conventional solution method, the SPE method facilitated the rapid and complete separation of the material with solution, which greatly reduced the time and labor cost. Compared with the commercial TiO₂ material, this new Ti⁴⁺-IMAC beads brought much more phosphopeptide identifications and much higher enrichment specificity.