The development of a unique buffered solvent system for characterization of phosphorylation sites at protein PTMs evolved from pH titration of assay buffer during chromatographic isolation of NM23-H1. As illustrated in , the single pH unit alterations performed caused substantial differences in peptide coverage and the number of successful peptide hits for both the control and auto-phosphorylated proteins. Only buffer systems at pH 5–6 successfully maintained the presence of the phosphate group on His118 for identification, though these pH levels did not provide optimum peptide coverage or the highest number of peptides mapped. Experiments at higher pH (pH 7.0 and pH 8.0) did not yield detection of peptide with the additional molecular weight of a phosphorylated histidine (866m/z (M+3H)), nor did they yield the non-phosphorylated ion (839m/z (M+3H)). The absence of a detected phosphorylation at the higher pH is likely due to the lower concentration of protons, and thus the inherent weaker ionization of this peptide species, rather than due to acid labile de-phosphorylation as expected at pH 3.0. A comparison of ions found at pH 3.0 (control), pH 5.0 (+ATP), and pH 6.0 (+ATP) are shown in .
Table 1 Liquid chromatography conditions and mass spectrometry results. The pH of the aqueous phase was adjusted in intervals of 1 pH unit from pH 8.0 to pH 2.5, the latter being our normal operating condition. A control (no ATP) and experimental (with ATP) auto-kinase (more ...)
Table 2 Corresponding b- and y-ions for phospho-peptide sequence. The peptide GDFC*IQVGRNIIH‡GSDSVESAEK, 866 m/z, is shown at pH 5.0 and pH 6.0. A corresponding nonphosphorylated peptide GDFC*IQVGRNIIHGSDSVESAEK, 839 m/z, from the pH 3.0 control was included (more ...)
NM23-H1 is shown to be auto-phosphorylated at precisely His118 (). Further, we verified phosphorylation of two other sites, Thr94 and Tyr53 (Supporting information
). As these are present in both the samples containing ATP and those without ATP (Supporting information
), they were likely phosphorylated in E. coli
prior to purification. Additionally, we also found that NM23-H1 in E. coli
acetylated Lys12 (Supporting information
Figure 1 Detection of histidine phosphorylation via LC/MS/MS. A and B) Fragmentation spectra of histidine phosphorylated peptide containing His118 from NM23-H1 in pH 5.0 and 6.0 solvents, respectively. Inlays represent zoomed regions showing appropriate fragment (more ...)
Histidine and aspartic acid phosphorylation events have not been well characterized due to their inherent pH sensitivity and the lack of phosphorylated His/Asp antibodies available commercially17
Whist others have employed a high pH solvent system to analyze pHis-containing peptides in histone H4, these data were obtained using milligram amounts of protein18, 19
. In the present work, we show that pH 5–6 is best to observe this PTM, using microgram quantities of NM23-H1. Higher pH reduces protein coverage for identification, as demonstrated in . In addition, this methodology allows for analysis of phosphorylations from whole cell lysates in a high-throughput manner, allowing for rapid characterizations of large numbers of proteins. This is beneficial for screening studies where the target proteins or phosphorylation sites of interest are unknown.
Presently, the NM23-H1 phosphorylation relay signaling pathway remains largely uncharacterized. Although it has been thought that most regulatory phosphorylations in eukaryotes occur on threonine, serine and tyrosine residues, this could be the result of failed characterizations of histidine and aspartic acid residue phosphorylations. Without methods to identify the latter, their absence in prior works could be the result of the inability to see them, rather than their lack of regulatory function. Here we show that the histidine/aspartic acid axis for protein regulation may incorporate the use of histidine phosphorylation for message transmittance.
Using this method, the role of NM23-H1 as a metastasis suppressor can be further evaluated. The presence of high concentrations NM23-H1 has been shown to be an indicator of prognosis in many cancers17, 20–27
, with its highest concentration in the non-tumorigenic state and a significantly reduced level in the metastatic state17
. Over-expression of NM23-H1 can eradicate tumor cell motility and invasion28
while promoting cellular differentiation, and shuts down anchorage-independent growth29
. The solvent system presented permits the detection of all five possible phosphorylation moieties (data not shown). Thus, these studies will significantly advance analysis of phosphorylation relay signaling in cellular regulation. Our goal is to employ this technique to define the phosphorylation relay pathway in cancer by mapping phosphorylation of all five possible phosphorylation residues, including histidine and aspartic acid residues. Based on the functional analysis of NM23-H1, these studies will provide unique insights into cellular regulation, cancer progression, and metastasis.
Presented is a novel buffer system showing histidine phosphorylation of NM23-H1, a putative human metastasis suppressor protein and possibly part of a two-component regulation in humans. Based on a pH titration of LC elution buffers and MS/MS identification, recombinant NM23-H1 is auto-phosphorylated and shows pHis118 at pH 5.0 and pH 6.0. The solvent system presented permits the detection of five possible phosphorylation moieties: serine, threonine, tyrosine, histidine and aspartic acid.