Enrichment techniques employed in phosphoproteomics

Abstract

Rapid changes of protein phosphorylation play a crucial role in the regulation of many cellular processes. Being post-translationally modified, phosphoproteins are often present in quite low abundance and tend to co-exist with their unphosphorylated isoform within the cell. To make their identification more practicable, the use of enrichment protocols is often required. The enrichment strategies can be performed either at the level of phosphoproteins or at the level of phosphopeptides. Both approaches have their advantages and disadvantages. Most enriching strategies are based on chemical modifications, affinity chromatography to capture peptides and proteins containing negatively charged phosphate groups onto a positively charged matrix, or immunoprecipitation by phospho-specific antibodies.In this article, the most up-to-date enrichment techniques are discussed, taking into account their optimization, and highlighting their advantages and disadvantages. Moreover, these methods are compared to each other, revealing their complementary nature in providing comprehensive coverage of the phosphoproteome.

Enrichment Strategies for Identification and Characterization of Phosphoproteome

“…The pH values in IMAC specificity are an important factor, because the acid dissociation constant (pK a ) of phosphate group at serine/threonine (pKa 2.1) and tyrosin residues (pK a 1.0) is lower than other acidic amino acid including glutamic acid (pKa 3.65) and aspartic acid (pKa 4.25). [53][54][55] To minimize these issues, loading and binding buffers during phosphopeptide enrichment with IMAC were often used to pH 2-2.5 containing with organic acid [e.g., trifluoroacetic acid (TFA), acetic acid, hydrochloric acid and formic acid]. 50 A variety of organic acids have been Immobilized metal ion affinity chromatography (IMAC) and (B) titanium dioxide (TiO 2 ) able for the specific enrichment of phosphopeptides from ordinary peptides used for high selectivity of IMAC, their influence resulted as follows: TFA > hydrochloric acid > formic acid > acetic acid.…”

“…50 A variety of organic acids have been Immobilized metal ion affinity chromatography (IMAC) and (B) titanium dioxide (TiO 2 ) able for the specific enrichment of phosphopeptides from ordinary peptides used for high selectivity of IMAC, their influence resulted as follows: TFA > hydrochloric acid > formic acid > acetic acid. 53 On the contrary, the elution of phosphopeptides bound on IMAC was performed at strong basic conditions containing ammonium hydroxide, urea, or tris-buffer. Although IMAC is a friendly used approach for enrichment of phosphopeptides, further optimization of loading and eluting buffers requires as an essential part.…”

“…[17][18][19][20][21] Capacity of acidic modifier in loading buffer for removing non-specific binding decreased as follows: DHB = salicylic acid = phthalic acid > benzoic acid = cyclohexanecarboxylic acid > phosphoric acid > TFA > acetic acid. 53 In recently, specificity of TiO 2 chromatography was optimized by adding glycerol 60 or citric acid. 61 On the other hand, the phosphopeptides bound onto TiO 2 resins was released by diverse amines and salts (e. g., ammonium bicarbonate, ammonium hydroxide, and pyrrolidine).…”

Advances in purification and separation of posttranslationally modified proteins

“…However, many commercially available anti-phosphotyrosine Ig antibodies also recognize phosphohistidine [64]. Immunoprecipitation is unsuitable for large-scale phosphoproteome studies as several parallel immunoprecipitation reactions are required for each phosphoamino acid, but it can be beneficial if specific phospho-site enrichment is desired [80]. Several derivatization-based phosphoprotein enrichment methods have been developed, but they involve several chemical reactions that are not 100 % efficient.…”

Special issue in quantitative mass spectrometric proteomics

“…Two separate review articles by Fila and Honys (2012) and Beltran and Cutillas (2012) published here discuss the plethora of methods to enrich and detect phosphopeptides and proteins. The numerous methods that have been developed to facilitate the detection of phosphorylation indicate how difficult it may be to accurately identify and measure this seemingly abundant PTM.…”