Citrate Boosts the Performance of Phosphopeptide Analysis by UPLC-ESI-MS/MS

Abstract

Incomplete recovery from the LC column is identified as a major cause for poor detection efficiency of phosphopeptides by LC-MS/MS. It is proposed that metal ions adsorbed on the stationary phase interact with the phosphate group of phosphopeptides via an ion-pairing mechanism related to IMAC (IMAC: immobilized metal ion affinity chromatography). This may result in their partial or even complete retention. Addition of phosphate, EDTA or citrate to the phosphopeptide sample was tested to overcome the detrimental phosphopeptide suppression during gradient LC-MS/MS analysis, while the standard solvent composition (water, acetonitrile, formic acid) of the LC system was left unchanged. With the use of UPLC, a citrate additive was found to be highly effective in increasing the phosphopeptide detection sensitivity. Addition of EDTA was found to be comparable with respect to sensitivity enhancement, but led to fast clogging and destruction of the spray needle and analytical columns due to precipitation. In contrast, a citrate additive is compatible with prolonged and stable routine operation. A 50 mM citrate additive was tested successfully for UPLC-MS analysis of a commercial four-component phosphopeptide mixture, a tryptic beta-casein digest, and several digests of the 140 kDa protein SETDB1. In this protein, 27 phosphorylation sites could be identified by UPLC-MS/MS using addition of citrate, including the detection of several phosphopeptides carrying 3-5 pSer/pThr residues, compared to identification of only 10 sites without citrate addition. A 50 mM citrate additive particularly increases the recovery of multiply phosphorylated peptides, thus, extending the scope of phosphopeptide analysis by LC-MS/MS.

Evaluation of column hardware on liquid chromatography–mass spectrometry of phosphorylated compounds

“…The selected metal ions were Al, Fe, Ti, Ni, Cr and Mo. Fe and Al can cause formation of chelating with phosphorylated compounds [17,19,[22][23][24]. The SUS316 were composed of Fe, Ti, Ni, Cr and Mo.…”

“…Third, we considered that the metal ions existing on S-S column inner surface were richer than GL-S column. The Fe and Al ions caused interaction with the phosphate groups [17,19,[22][23][24]. The S-S column was made from SUS316 which included 50% or more of iron.…”

“…[11]. In order to overcome this drawback, three methods of improvement could be used; (1) deactivation of chromatographic system such as valves, needles and tubes [9,[12][13][14][15]; (2) addition of chelate reagents in pretreatment and sample solvent [11,[16][17][18]; (3) using alkaline mobile phase as in immobilized metal affinity chromatography [16,19]. The improvements with methods (1) and (2) were due to prevention from interaction between phosphate compounds and metal ions on the surface of flow path.…”

Affinity and Chemical Enrichment for Mass Spectrometry-Based Proteomics Analyses

“…These carriers prevent not only general loss of peptides to surfaces, but also phosphopeptide-specific losses which are thought to occur on metal oxide surfaces encountered throughout the LC system and autosampler, or on trace metal ions immobilized on reversed-phase resins typically used to separate phosphopeptides prior to or online with mass spectrometry analysis. These interactions may be minimized by addition of EDTA or citrate to the elution buffer (Winter et al, 2008).…”

Recent advances in micro-scale and nano-scale high-performance liquid-phase chromatography for proteome research

“…According to Giddings' theory [11], resolution, efficiency, and throughput in 1D micro-or nano-HPLC benefit from reduced particle size. However, such changes can result in increased back pressure, which has accelerated the development of ultraperformance liquid chromatography (UPLC) [12][13][14].…”