Protein sequencer maintenance and troubleshooting.

Abstract

Modern automated sequencers, both gas-phase and pulsed-liquid forms, are normally efficient and reliable; this chapter is intended for use when they are not. The author’s own experiences are almost wholly concerned with ABI (Applied Biosystems) instruments; however, the principles described will apply to most other manufacturers’ sequencers. This chapter will address the various functions individually, in an attempt to identify typical faults and remedies as outlined in Table 1 and Notes 1–15. Table 1 A Trouble-Shooting Guide for Protein Sequencers 1. Blank chromatograms-possible failure of solvent and reagent delivery (see Section 3.1.) or of PTH a a transfer (see Sections 3.4. to 3.6., and Note 2). 2. Erratic retention times on the PTH a a. chromatogram, (see Section 3.6. and Note 4). 3. New peak appearing in every PTH a a. chromatogram, close to proline (see Note 4). 4. Compacting of the hydrophobic region of the PTH a.a. separation; most noticeable on the PTH a.a. standard; can also be accompanied by peak broadening (see Note 5). 5. Increased carry over or “lag” from cycle to cycle (see Section 3.2. and Note 6). 6. Abnormally high levels of DPTU and DPU (see Sections 3.2. and 3.3. and Note 7). 7. Excessive background peaks on PTH a∶a. chromatograms, starting within the early stages of a run (see Section 3.2. and Note 8). 8. Low repetitive yield i.e., less than 90–92% (see Section 3.2. and Note 9). 9. Low yield of PTH-ser or thr, or generally low yield-due to over- or underdrying in conversion flask (see Section 4). 10. Inability to reach the C-terminus of peptides (see Note 10). 11. Artifact peaks from the addition of DTT (see Note 11). 12. Sloping baseline at the start of the PTH separation and abysmal resolution of the early-fluting amino acids (see Note 12). 13. A very concave “smiling baseline” on PTH chromatograms (see Note 13). 14. High pressure indicated on analyzer display; usually first noticed as a high pressure shutdown during a solvent purge (see Note 14). 15. Huge, off-scale peaks on the PTH a∶a. chromatograms (see Note 15).