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Protein Identification

Protein/Peptide Microsequencing (Beckman)

Electrospray Mass Spectrometry

Porton Instruments 2020 Information

Sequencing employs a Porton Instruments 2020 sequencer with on-line Beckman Gold HPLC system and Beckman 32karat analysis system. This instrument utilizes Edman chemistry to determine the primary structure of proteins by N-terminal sequence degradation. The sequencer removes the N-terminal amino acid and converts it to the PTH derivative while leaving the remainder of the chain intact. In subsequent cycles, the newly exposed N-terminal amino acids are removed and delivered to the analytical system as PTH derivatives for amino acid identification.

Sequencing can be performed on tens of picomoles of protein or peptide under optimal conditions. However, the number of residues sequenced on a given sample can vary significantly depending on the nature, amount, and purity of that sample. Therefore, you should target approximately 100-200 pmol of protein/peptide for sequence analysis. The more sample there is to sequence the better the chances for a successful analysis.

There is a minimum fee for a "sequencability check" of 4 cycles. Essentially, the first 4 cycles will be monitored and if it appears that the sample is blocked, contaminated, or of insufficient quantity or quality to obtain useful information, the run will be aborted. There is a basic summary of some of the practical considerations involved in isolating a protein for N-terminal sequence analysis by electroblotting onto PVDF membrane. If this information is of interest, request a copy or find it on our protocol web page.

DECA XP+ Mass Spectrometer Information

The facility has a Thermo Electron DECA XP+ electrospray ionization mass spectrometer (ESI MS) in line with a Surveyer liquid chromatography (LC) system and autosampler. The instrument is used for protein/peptide identification from an enzymatic digest on less than 1 pmol of sample. A protocol of in-gel tryptic digest of a protein band is available on our website.

Briefly, a peptide digest is injected via HPLC onto a capillary column for nanospray ionization. The peptides of the digest are separated on the reverse phase column, plus ionized to individual ion droplets at the tip of the column. The peptide ions at the acidic PH of the mobile phase are positively charged, and are introduced into the ion trap in positive ion mode by adjusting voltage. A vacuum allows the ions to move unimpeded. Ions are "trapped" with a combination of voltage frequency adjustments and then ions of a specific mass-to-charge ratio (m/z) are allowed to scan out and be detected as (MS) data or remain "trapped". Trapped ions can be further subjected to collisions with gas molecules and a change in radio frequency to produce "collision-induced" dissociation of the trapped peptide ions. The fragments produced ("daughter ions") can then be ejected from the trap to produce the MS/MS spectrum.

The MS and MS/MS data can then be used to search databases for identification, and often detect the exact sequence location of modifications on the peptide (eg: phosphorylation sites and researcher-induced modifications).