LC/MS and LC/MS/MS determination of protein tryptic digests

Abstract

Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray (ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome c, bovine β-lactoglobulin A, and bovine β-lactoglobulin B.

Application and Challenges in Using LC–MS Assays for Absolute Quantitative Analysis of Therapeutic Proteins in Drug Discovery

“…As early as the late 1980's, Henion's laboratory explored the application of microflow in the quantification of protein tryptic digests [95]. The microflow UHPLC technologies have been applied in the ana lysis of small molecules [96,97] and should provide promising advantages in the protein quantification in the biological matrices as the technologies advance.…”

Strategies for improving sensitivity and selectivity for the quantitation of biotherapeutics in biological matrix using LC-MS/MS

“…As described, the quantitation for larger therapeutic proteins (molecular weight greater than 5 kDa) is based on the approach of enzymatic digestion followed by monitoring one or more of the proteolytic peptides [7,12,13]. As illustrated in FIGURE 1, the simplest bioanalytical workflow includes the following basic steps: enzymatic digestion to generate surrogate peptides (one or more peptides to unambiguously represent the entire protein); sample clean up of the digested samples; chromatographic separation and MS/MS detection.…”

“…Instead of detecting intact proteins, contemporary quantitation of protein with MS relies on detecting a surrogate peptide (a unique peptide to represent the entire protein) released from the target protein based on an enzymatic digestion model [7]. To improve specificity and selectivity of the mass spectrometric detection, quantitation experiments are performed by coupling liquid chromatography with mass spectrometry (i.e., LC-MS/MS).…”

MS/MS applications in biological problems

“…As soon as ES ionization allowed the HPLC/MS coupling, ESI of peptides generated by digestion of proteins became an attractive strategy for the determination of protein sequences (69). In a study by Griffin et ul.…”