Analysis of Protein Phosphorylation by Capillary Liquid Chromatography Coupled to Element Mass Spectrometry with ³¹P Detection and to Electrospray Mass Spectrometry

Abstract

A method for phosphopeptide identification by capillary liquid chromatography (muLC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described. ICPMS is used for 31P detection and ESI-MS provides the corresponding molecular weight information. Alignment of the two separate muLC runs is performed using the baseline distortion at the elution front, which shows up in both muLC-ICPMS and muLC-ESI-MS. Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP. The detection limit achieved for the muLC-ICP-HRMS runs is approximately 0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins. These include beta-casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit. The tryptic phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs. The two most important aspects of using muLC-ICPMS with 31P detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of 31P in the muLC eluate. Thus, muLC-ICPMS with 31P detection is introduced as a new, robust, and specific method in phosphoproteomics.

Determination of the relative energies of activation for the dissociation of aromatic versus aliphatic phosphopeptides by ESI-FTICR-MS and IRMPD

“…Several methods of phosphopeptide mapping have also been proposed, taking advantage of the relative speed, sensitivity, and adaptability of MS. These methods include the use of phosphatases [17][18][19][20], 31 P monitoring by inductively couple plasma (ICP)-MS [21], collision induced dissociation (CID) [22][23][24][25], precursor ion scanning [24, 26 -31], neutral loss scanning [32], nozzle-skimmer dissociation [33,34], metastable decomposition [35][36][37], and electron capture [38].…”

Inductively Coupled Plasma–Mass Spectrometry (ICP-MS) for Quantitative Analysis in Environmental and Life Sciences: A Review of Challenges, Solutions, and Trends

“…Plasma instabilities until its extinction, carbon deposition on the cones and lens system, or signal suppression are wellknown further detrimental effects related to the introduction of high amounts of organic solvents into an ICP, which often limits the hyphenation of standard reversed-phase (RP) HPLC setups with ICP-MS detection, in particular when working with solvent flow rates between 0.1 and 1 mL min -1 . Membrane desolvation, 41 oxygen addition, 42 spray chamber temperatures below 0 8C, 20 or reduced injector tubing inner diameters 42 are often-used tools to minimize the organic load of the plasma, in particular when utilizing RP-HPLC conditions, which are necessary for many ICP-MS, as well as the development of collision/reaction cells, during the last decade has minimized the interference problem that naturally delimits the accurate detection and quantification of many elements. Meanwhile, most elements of the periodic table including metals, metalloids, semimetals, non-metals, or halogens can be accurately determined by ICP-MS, allowing their application as surrogate standards for the quantification of all kinds of molecules whenever the stoichiometry of the used element tag is known.…”

“…However, incomplete peptide recovery from the desolvation systems has been observed, especially for late eluting compounds. 41 A further paper focused on the demonstration of phosphorylation degree determination of different model proteins via simultaneous measurement of the phosphorus and sulfur content of the eluting peptide fractions. 44 Minimized dead volumes are essential for highly resolved nano or capillary LC separation of phosphopeptides.…”

State-of-the-art in phosphoproteomics

“…The only method capable of accurate quantitation of phosphorylations without the need for stable isotope labels is element MS such as ICP-MS [95,96]. This technique may be used for secure identification of phosphorus-containing proteins and peptides.…”