Post-source decay peptide sequencing

Heilman, U. and Bhikhabhai, R. (2002) Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer equipped with a variable voltage reflector. Rapid Commun. Mass Spectrom., 16, 1851-1859. 

Both collisional activation (in ion traps) and post source decay (in curved field reflectron TOF analyzers) have been used successfully to obtain sequence ions from peptides prepared in situ on the sample holder. Single Dalton mass windows are advantageous for precursor selection, as are realized in ion-trap and trap-TOF configurations. Publicly available search algorithms can be used... 

Cordero, M.M. Cornish, T.J. Cotter, R.J. Lys, LA. Sequencing Peptides Without Scanning the Reflectron Post-Source Decay With a Curved-Field Reflectron TOF Spectrometer. Rapid Commun. Mass Spectrom. 1995, 9, 1356-1361. 

Peptide sequencing via post-source decay and tandem mass spectrometry. 339... 

Chen, P., Nie, S., Mi, W Wang, X.-C., and Liang S.-P. (2004) De novo sequencing of tryptic peptides sulfonated by 4-sulfophenyl isothiocyanate for unambiguous protein identification using post-source decay matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 18,191-198. 

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. 

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... 

Figure 4. Tryptic peptide digest spectrum of spot 6 and post source decay spectrum of a tryptic peptide (T8), MYSPTSILDIR, with mass at m/z 1295.94 from the protein at spot 6. (A) MALDI reflector mass spectrum of tryptic peptide mixture derived from spot 6. (B) Mass spectrometric determination of partial peptide sequence of a tryptic peptide (T8). Y" series ions were observed.

Figure 6. Post source decay spectrum of N-terminal tryptic peptide, PIVQNIQGQMVHQAISPR, of HfV-1 p24 . a-, b-, and y"-dominant fiagment ions, defined according to the nomenclature of Biemann (1990), are shown, confirmhig the partial sequences (amino acids 1-7, and amino acids 10-18).fPis defined as a formylated prohne.

A certain amonnt of structural information, snch as sequencing of peptides, can be obtained from single-analyzer MALDl-TOF systems used in the reflectron mode becanse some ions leaving the ion source are metastable (Section 3.3.1.2) and fragment as they move down the flight tube the process is called post-source decay (PSD). The technique is limited because it is not possible to preselect ions for fragmentation and becanse stepwise changes in the parameters of the reflectron are needed to obtain prodnct ion spectra. 

Cordero MM, Cornish TJ, Cotter RJ, and Lys lA (1995) Sequencing peptides without scanning the reflectron post-source decay with a curved-field reflectron time-of-flight mass spectrometer. Rapid Communications in Mass Spectrometry 9 1356-1361. 

Marekov, L. N., Steinert, P. M. (2003) Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser des-... 

Wang, D., Kalb, S. R., Cotter, R. J. (2004) Improved procedures for N-terminal sulfonation of peptides for matrix-assisted laser desorption/ionization post-source decay peptide sequencing. Rapid Commun Mass Spectrom, 18,96-102. 

In-Source Decay (ISD). In the next chapter we will describe a method for amino acid sequencing of peptides on reflectron TOF instruments known as post-source decay (PSD). This method exploits the considerable metastable fragmentation that occurs in the drift region after the ions are extracted from the ion source. The resulting product ions spend a shorter time in the reflectron, and therefore arrive at the detector sooner than their precursors. 

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