Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts

Abstract

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used successfully to detect phosphorylation sites in proteins. Applications may be limited by the low response of phosphopeptides compared to nonphosphorylated peptides in MALDI MS. The addition of ammonium salts to the matrix/analyte solution substantially enhances the signal for phosphopeptides. In examples shown for equimolar mixtures, the phosphorylated peptide peaks become the largest peaks in the spectrum upon ammonium ion addition. This can allow for the identification of phosphopeptides in an unfractionated proteolytic digestion mixture. Sufficient numbers of protonated phosphopeptides can be generated such that they can be subjected to postsource decay analysis, in order to confirm the number of phosphate groups present. The approach works well with the common MALDI matrices such as alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, and with ammonium salts such as diammonium citrate and ammonium acetate.

Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification

“…In recent years, mass spectrometry has become a key technology for characterization of protein phosphorylation and phosphoproteome analysis. Two complementary ionization techniques, MALDI and ESI, in combination with a variety of mass analyzers, have been used to identify phosphopeptides and determine the phosphorylated amino acids on the peptides (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). In most cases, characterization of phosphopeptides by MS requires selection of phosphorylated peptides from complex peptide mixtures resulting from proteolysis of phosphorylated proteins followed by MS/MS to confirm the phosphorylation and to identify the phosphorylated amino acid residues on phosphopeptides containing more than a single serine, threonine, or tyrosine residue.…”

Recombinant Brassinosteroid Insensitive 1 Receptor-Like Kinase Autophosphorylates on Serine and Threonine Residues and Phosphorylates a Conserved Peptide Motif in Vitro

“…MALDI-MS is a powerful technique for precise M r determination of a complex mixture of peptides. When diammonium citrate is included in the MALDI matrix, the peak intensity of phosphorylated peptides is increased relative to unphosphorylated peptides (Asara and Allison, 1999). Moreover, when the amino acid sequence of the protein is known, the mass of each potential tryptic peptide can be calculated and the phosphorylation state of a specific peptide can then be assigned from mass-to-charge ratio (m/z) values in MALDI-MS spectra.…”

“…Fractions were collected by hand and 1 L of each fraction was checked for 32 P incorporation using a phosphor imager. The radioactive fractions were then analyzed by MALDI-MS to determine the sites of phosphorylation as previously described (Asara and Allison, 1999).…”

MALDI Mass Spectrometry Using 2,4,6-Trihydroxyacetophenone and 2,4-Dihydroxyacetophenone with Cyclodextrins: Suppression of Matrix-related Ions in Low-molecular-weight Region

“…To overcome such drawbacks, many attempts have been made so far, including the use of sugars, 3 ammonium salts, 4,5 phosphoric acid, 6 serine, 7 and proteins 8 with conventional organic MALDI matrices. Such matrix-free techniques as the desorption/ionization on silicon (DIOS) 9 and the use of a nano-structured surface 10 have been proposed as well.…”