Solid‐phase derivatization of tryptic peptides for rapid protein identification by matrix‐assisted laser desorption/ionization mass spectrometry

Abstract

Solid-phase sulfonation of tryptic peptides adsorbed to C 18 mZipTips 2 has been carried out to facilitate de novo sequencing with mass spectrometry. Peptides are reacted with the sulfonation reagent while they are still adsorbed to the solid phase. Excess reagent passes through the ZipTip 2 to waste. Washing the products before subsequent elution from the mini-column also affords sample cleanup prior to analysis. Near quantitative N-terminal sulfonation can be achieved reliably at room temperature in only a few seconds. The method has been applied successfully to model peptides and to solution or in-gel digests of proteins. Current sequencing limits are about 100 fmol of protein.Multiplexed sample sulfonation reactions have been carried out with a manual 8-position micropipettor or using centrifugal force to reliably pass reagents and wash solutions over sampleloaded ZipTips 2 . With multiplexing, overall preparation times have been reduced to about 1 min per sample. The solid-phase format facilitates efficient use of precious digest samples by enabling them to be recovered from the matrix-assisted laser desorption/ionization (MALDI) sample stage after mass fingerprinting, derivatized and re-analyzed by MALDI postsource decay mass spectrometry. Copyright # 2002 John Wiley & Sons, Ltd.Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is widely used for the identification of proteins that have been separated by 1D-or 2D-gel electrophoresis. Typically, gel-separated proteins are digested with trypsin, 1 and the resulting mixtures of tryptic peptides are analyzed by MALDI-MS. The measured masses of the tryptic peptides are compared to theoretical peptide masses calculated for each protein in various sequence databases. The resulting lists of candidate proteins that most closely match the input data are rank ordered using various scoring algorithms. 2±6 In favorable cases definitive protein identifications are possible using this simple`peptide mass fingerprinting' approach. 7 Unfortunately, peptide mass fingerprinting can also lead to ambiguous protein identifications. This may result because of insufficient protein sequence coverage. Limited sequence coverage is often observed at low analyte levels because of poor recovery of tryptic peptides from the gels and because of variable peptide response under MALDI conditions. It is also known that Lysterminated tryptic peptides are less responsive than Arg-terminated tryptic peptides. 8 Furthermore, co-migrating proteins may produce complex mixtures of tryptic peptides that are difficult to de-convolute and interpret. Finally, the sequences of some analyte proteins are novel, so their experimentally observed tryptic mass fingerprints cannot be matched with confidence to any entries in the databases.Complementary tandem mass spectrometry methods are often used to sequence tryptic peptides to resolve ambiguous protein identifications resulting from MALDI mass fingerprinting. The discriminating power of peptide sequence data is extremely high. Defin...

TSG-6 Transfers Proteins between Glycosaminoglycans via a Ser28-mediated Covalent Catalytic Mechanism

“…4, A-D) (36). The CAF protocol simplifies spectra interpretation because y-ions are favored, although in this case the presence of two peptides complicated the spectra due to the increased number of possible fragment ions (supplemental data Tables S1 and S2).…”

Charge derivatization by 4‐sulfophenyl isothiocyanate enhances peptide sequencing by post‐source decay matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

“…21 In this case the addition of sulfonic acid groups to the N-termini of tryptic peptides facilitates de novo peptide sequencing by PSD MALDI-TOFMS. These derivatives promote efficient charge-site initiated cleavage of backbone amide bonds and they allow the selective detection of only a single series of fragment ions that contain the original C-terminus of the peptide (y-, y 17 ions).…”

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

“…These reagents are commercially available and allow direct sulfonation of subpicomol quantities of tryptic peptides. Later, the method was refined by using a water-soluble, sulfopropionic acid NHS-ester [18], a reagent that recently became available in a commercial kit (Ettan CAF MALDI sequencing kit, Amersham Biosciences, Uppsala, Sweden) [19]. Very recently, a similar derivatization procedure was reported using 4-sulfophenyl isothiocyanate as the derivatization reagent [20,21].…”