C-terminal ladder sequencing

Thiede, B., Salnikow, J. and WittmannLiebold, B. (1997) C-terminal ladder sequencing by an approach combining chemical degradation with analysis by matrix-assisted-laser-desorption ionization mass spectrometry. Eur. J. Biochem., 244 (3), 750 4. 

Analysis of the mixture using matrix-assisted desorption ionization mass spectrometry (section 5.3) allows for direct sequence determination from the snccessive mass differences of the peptide ladder. The application of the volatile trifluoroethyl isothiocyanate results in a significant optimization of this procedure and allows for peptide sequencing at the femtomole level (Bartlet-Jones et al., 1994). C-terminal ladder sequencing uses ammonium thiocyanate in acetic anhydride coupled with mass spectrometric analysis of truncated peptides (Thiede et al, 1997). Matrix-assisted desorption ionization instruments with delayed extraction (Brown and Leimon, 1995) allow for the discrimination of aU amino acids, except Leu and He. 

D. H. Patterson, G. E. Tarr, F. E. Ragnier, and S. A. Martin, C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with car-boxypeptidase Y time-dependent and concentration-dependent digestions. Anal. Chem. 67, 3971-3978 (1995). 

Fig. 1. Core histone modifications. Human histone N-terminal and in some cases C-terminal amino acid sequences are shown. The modifications include methylation (M), acetylation (Ac), phosphorylation (P), ubiquitination (U), and ADP ribosylation (step ladder). The sites of trypsin digestion of histones in nucleosomes are indicated (T).

Top) peptide ladder sequencing principle. Phenyl isothiocyanate (PITC) produces phenylthiohydantoin (PTH) of the terminal amino acid and a new peptide with one less amino acid. Phenyl isocyanate (PIC), in low quantity, produces N-terminal phenylcarba-mate (PC) from a small fraction of each peptide. (Bottom) example of sequencing of [Glu1]fibrinopeptide B. Reproduced (modified) from Chait B.T., Wang R., Beavis R.C. and Kent S.B.H., Science, 262, 89, 1993, with permission. 

In the early LC-MS-MS studies, CID is performed with low-energy collisions by means of triple-quadrapole instruments. Under these conditions, series of N-terminal b-ions and C-terminal y-ions result from cleavages at the peptide bond and charge retention on either side, although double-charge tryptic peptide ions tend to favour fragmentation towards more abimdant y-ions. From these ladders of sequence ions in the MS-MS spectrum, the amino-acid sequence of the peptide may be derived. This is the bottom-up protein identification approach (Ch. 18.3.1). 

The de novo method of sequencing that uses enzymes to digest the terminal amino acids from a peptide is called peptide ladder sequencing. In this technique, an enzyme such as carboxypetidase-Y is used to digest one amino acid at a time from the C-terminus. No additional fragment peaks are formed. This method, unlike the MS-MS de novo technique, requires that the peptide be pure. In addition to the need for a pure peptide, another drawback of this technique is the somewhat lengthy sample preparation. The major advantage of this technique is the ease with which the mass spectrum can be interpreted and the partial sequence determined. 

My recommendation protect the N-terminal ends during purification. But what if the N-terminus is already blocked You can try sequencing C-terminally (see Section 7.6.5) or with the mass spectrometer (see Section 7.6.6). If you cannot do this or do not want to, you have no choice but to give up and cleave the protein into peptides, and separate and sequence them. Which of the three methods is to be preferred depends on the local climate. If you can find someone in the institute who knows a lot about sequencing by mass spectrometer, enlist the services of this person. For one or a few sequences it is not worth studying the peptide ladder technique for yourself (see Section 7.6.6), especially if you have to purchase a mass spectrometer first. 

H., and Kang, C. (2001) DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry. Nucleic Acids Res., 29 (3), Ell. 

The ability of FAB mass spectra to deliver peptide sequence information was soon recognized [15,130]. Initially, the sequence was derived from fragment ions observed in the full scan spectra [15,96]. Another approach to sequence information is to subject the peptide to enzymatic hydrolysis by a mixture of several carboxy-peptidases to produce a series of truncated molecules. The FAB spectrum of the mixture then reveals the C-terminal sequence [131,132]. In the MALDI community, this approach became known as peptide ladder sequencing [133]. 

Many structural biology and biochemistry studies rely on the precise identification of the N-/C-terminal sequence of a protein (e.g., signal peptide identification, determining the cleavage site and specificity of a novel protease, etc.). Digestion of proteins in arrays of carboxypeptidase Y at different concentrations with MALDl-MS-based readout can be employed to derive C-terminal sequence information for proteins. The amino-terminus of a protein can be derived by wet chemistries similar to Edman degradation coupled to MS-readout. This method of protein ladder sequencing, reported first by Kent and... 

Fig. 4. Primer extension analysis of rRNAs isolated from four plant species treated with dimethyl sulfate. Sequencing lanes are indicated as A, T, G, and C. A plus sign (+) over the lane denotes RNA prepared from DMS-treated tissues a minus (—) denotes RNA isolated from mock-treated tissues. The nucleotide numbers are the coordinates at which primer extension reactions terminate (see Figs- 3 and 5A). The position of the modified base in the sequence of the 18 S rRNA is determined by comparing novel bands in the DMS-reacted RNA with the sequencing ladder and adding one nucleotide (see Fig. 2 for details). Various regions of the RNA are shown for the different plant species. Several sites of chain termination arising from nascent structure are also evident.

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