Songyun Xu

Capture of Phosphopeptides Using α-Zirconium Phosphate Nanoplatelets

Alpha-zirconium phosphate nanoplatelets (alpha-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of alpha-zirconium phosphate were incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice with water followed by an aqueous solution of 80% acetonitrile to form the binding agent. Alpha-ZrPN were able specifically to capture phosphoserine-containing peptides from a tryptic digest of a complex peptide mixture in which its abundance was only 0.05%. Alpha-ZrPN also bound peptides containing phosphothreonine and phosphotyrosine. The limit of detection for phosphopeptides is approximately 2 fmol, based on using matrix-assisted laser desorption/ionization mass spectrometry. Alpha-ZrPN were applied for the analysis of tryptic digests of mouse liver and leukemia cell phosphoproteomes and succeeded in identifying 158 phosphopeptides (209 phosphorylation sites) from 101 phosphoproteins in mouse liver lysate and 78 phosphopeptides (104 phosphorylation sites) from 59 phosphoproteins in leukemia cell extract. For these two tryptic digests, the alpha-ZrPN approach is able to capture more phosphopeptides than that obtained from TiO2 particles or from Fe(3+)-IMAC beads, but each method is able to bind some phosphopeptides that the others do not.

Monitoring enzyme reaction and screening enzyme inhibitor based on MALDI-TOF-MS platform with a matrix of oxidized carbon nanotubes

A matrix assisted laser desorption/ionization time-of-flight mass spectrometry platform for quantitatively monitoring enzyme activity and screening enzyme inhibitors has been demonstrated. The described method employs a new matrix of oxidized carbon nanotubes. Compared with the traditional fluorescence approach, this label-free method has the advantage of directly identifying the substrates and products in enzymatic reactions. Moreover, the method could be conveniently carried out with any commercial mass spectrometer without modification. We quantitatively monitored the acetylcholinesterase activity and screened acetylcholinesterase inhibitors with a detection rate of about 3.3 s per sample.

A matrix of 3,4-diaminobenzophenone for the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A new matrix of 3,4-diaminobenzophenone (DABP) was demonstrated to be advantageous in the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With DABP as a matrix, intact oligonucleotide ions can be readily produced with lower laser powers, resulting in better detection limits, less fragmentation and fewer alkali metal ion adducts compared with the results obtained with conventional matrices. Importantly, minimal fragmentation and fewer alkali metal ion adducts were seen even at low concentrations of oligonucleotides. It was also found that samples prepared with DABP are highly homogenous and therefore reducing the need for finding ‘sweet’ spots in MALDI. In addition, excellent shot-to-shot reproducibility, resolution and signal-to-noise ratio were seen with DABP as the matrix.

3,4‐Diaminobenzophenone Matrix for Analysis of Oligonucleotides by MALDI‐TOF Mass Spectrometry

This unit contains procedures for analysis of oligonucleotides by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry with 3,4‐diaminobenzophenone (DABP) as a matrix. This new matrix has demonstrated advantages in the analysis of oligonucleotides. With DABP as a matrix, intact oligonucleotide ions can be readily produced with lower laser powers, resulting in better detection limits, less fragmentation, and fewer alkali‐metal ion adducts compared with results obtained using conventional matrices. Minimal fragmentation and fewer alkali‐metal ion adducts were seen even at low oligonucleotide concentrations. It was also found that samples prepared with DABP are highly homogenous, therefore reducing the need to find “sweet spots” in MALDI. Finally, excellent shot‐to‐shot reproducibility, resolution, and signal‐to‐noise ratio are observed using the DABP matrix. Curr. Protoc. Nucleic Acid Chem. 31:10.12.1‐10.12.6.