Post-source decay MALDI

Some Examples. Earlier in this chapter, we discussed the isolation and identification of an antigen AMAPBTLLL bound to a novel murine Class IB MHC molecule Qa-1. In that example, the amino acid sequence was determined from the MALDI mass spectra of ladder peptides obtained from aminopeptidase M, carboxypeptidase P, and chymotrypsin digestions. As shown in Figure 10.19, the amino acid sequence can also be determined from the post-source decay MALDI mass spectrum of the intact peptide. In this case, a curved-field reflectron was used, so that the entire mass spectrum was recorded without changing the reflectron voltage. 

Tsarbopoulos, A., Bahr, U., Pramanik, B.N. and Karas, M., Glycoprotein analysis by delayed extraction and post-source decay MALDI-TOF-MS. Int. J. Mass Spectrom. Ion Processes, 169/170, 251-261 (1997). 

Experimentation showed that the protein was not glycosylated and that the sequence at the iV-amino acid terminus corresponded to that expected. The C-terminus sequence, however, did not correspond to that predicted and these data were interpreted in terms of the presence of a heterogeneous, truncated, protein. A study of the tryptic digest fragments from this protein with matrix-assisted laser desorption ionization (MALDI) with post-source decay enabled the authors to suggest the positions at which the parent protein had been truncated. 

Figure 6 Partial MALDI-TOF mass spectrum from PMMA generated by ATRP with ethyl-2-bromoisobutyrate as initiator (inset shows theoretical isotope distribution for lithiated 18-mer of structure 3). (Peak labelled 3 arises from post-source decay of 3 [10].)...

The development glycopeptide libraries obtained by the split-mix method is severely hampered by the lack of concurrent development of a general, facile separation and characterization technology. Some headway has been made with chemical coding of the libraries, but very few direct methods of analysis exist. One promising method that could be applied to the direct characterization of both types of libraries is mass spectrometry. More specifically, post-source-decay matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (PSD-MALDI-TOF-MS) and CID-FAB/MS/MS have been used to characterize glycopeptides.53-55... 

Several original papers must be mentioned that deal with mass spectrometric techniques which the numerous reviews do not comprise. Kaufmann and coworkers268,288 studied the mass spectrometric analysis of carotenoids and some of their fatty acid esters using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and its post-source-decay (PSD) variant. Some advantages concerning the thermal instability and limited solubility were discussed, but the fragmentation paths of the carotenoid cations were found to be essentially the same as those observed with conventional techniques. 

Note In particular the MALDI-TOF community has coined some sort of an own terminology, e.g., in-source decay (ISD) for all fragmentations occurring within the ion source, post-source decay (PSD) instead of metastable ion dissociation and fragment analysis and structural TOF (FAST) for the specific mode of operation of a ReTOF to detect metastable ions. 

Kaufmann, R., Wingerath, T., Kirsch, D., Stahl, W., and Sies, H. 1996. Analysis of carotenoids and carotenol fatty acid esters by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source-decay mass spectrometry. Anal. Biochem. 238 117-128. 

Kaufmann, R., Kirsch, D., and Spengler, B. (1994). Sequencing of peptides in a time-of flight mass spectrometer Evaluation of post source decay following matrix-assisted laser desorption ionization (MALDI). Int. ]. Mass. Spectrom. Ion Processes 131, 355-385. 

B. SPENGLER, D. KIRSCH and R. KAUFMANN obtain structural information with reflectron TOF mass spectrometry (MALDI post-source decay) [66],... 

With the advent of very sensitive ionization techniques such as matrix assisted laser desorption (MALDI) coupled with time-of-flight (TOF) mass analysis, measurement of the intact mass of peptides at sub-pmol levels has become a reality (2) of which we have taken advantage for the systematic screening of HPLC fractions. Partial sequence information can be obtained by carrying out enzymatic hydrolysis with exoproteinases (e.g. carboxypeptidases and aminopeptidases) (3, 4). More recently, MALDI has been used to measure metastable decomposition occurring in the first field free region of a reflectron TOF instrument (referred to as post source decay (PSD)) with only marginally more sample (5-7). 

We have implemented scanning methodologies using MALDI-TOF mass spectrometry to partially purified venom from C. striatus and C. ermineus. We have carried out specific derivatizations in order to deduce composition and sequence information. Together with an intact mass these measurements are used to determine whether an ionized species observed in the MALDI mass spectrum corresponds with the intact protonated molecule of a previously characterized conotoxin. The information obtained from derivatizations is also important when the ionized species does not correspond with the intact mass of peptides of known sequence. In that case, post source decay of the native and derivatized species may help assign the fragment ions. 

Recently, fragmentations by post source decay (PSD) was introduced as a technique to obtain structural information in MALDI MS [6,7]. We describe here the combined use of automated Edman sequencing and MALDI sequencing for the determination of proteolytic peptide fragments in the low picomole range. 

A 2 pmole quantity of the 62-kDa protein was digested in situ with trypsin in an excised polyacrylamide gel slice. The resulting peptides were resolved by reversed-phased HPLC. Peaks detected by HPLC were selected for further analysis by sequencing, LC-MS, and MALDI-TOF. One peak with a retention time at approximately 30.25 failed to yield an interpretable sequence. Upon further observation, the mass observed by LD-MS was consistent rvith the N-terminal residues of the 62-kDa protein with the addition of an N-terminal acetyl group. Post source decay analysis revealed that this peptide was indeed the predicted amino terminal tryptic peptide (Figure 3). All other peptide peaks matched various internal sequences of the r62-kDa protein. 

In this study, we used matrix-assisted laser desorption ionization /Mass Spectrometry (MALDI/MS) to identify the peptides released from gastric parietal cell microsomes. MALDI, because of its sensitivity and relative tolerance to the presence of salts and buffers was examined for the analysis of unfractionated proteolytic digests (9, 10). MALDI with post-source decay (PSD) analysis was used to obtain sequence information on peptides even in crude digestion mixtures. Our strategy (Figure 1) consisted of proteolysis of intact vesicles, centrifugation at high speeds to separate membrane bound and soluble fractions and analysis of the mixture of released peptides by MALDI/MS. In addition, to increase the... 

B. Spengler, Post-source decay analysis in MALDI-MS of biomolecules, J. Mass Spectrom., 32 (1997) 1019. 

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