Diagnostic protein discovery using proteolytic peptide targeting and identification

Abstract

Plasma protein profiling with mass spectrometry is currently being evaluated as a diagnostic tool for cancer and other diseases. These experiments consist of three steps: plasma protein fractionation, analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and comparisons of the MALDI profiles to develop diagnostic fingerprints using bioinformatic techniques. While preliminary results appear promising in small sample groups, the method is limited by the sensitivity of MALDI-MS for intact proteins, the limited mass range of MALDI-MS, and difficulties associated with isolating individual proteins for identification to validate the diagnostic fingerprint. Here we present an alternative and improved method directed toward diagnostic protein discovery, which incorporates proteolytic peptide profiling, bioinformatic targeting of ion signals, and MALDI tandem mass spectrometry (MS/MS) peptide sequencing, rather than fingerprinting. Pancreatic cancer patients, pancreatitis patients, and controls are used as the model system. Profiling peptides after enzymatic digestion improves sensitivity and extends the accessible protein molecular weight range when compared to intact protein profiling. The first step is to extract and fractionate the proteins from plasma. Each fraction is digested with trypsin and subsequently analyzed by MALDI-MS. Rather than using bioinformatic analysis as a pattern-matching technique, peptides are targeted based on the disease to control peak intensity ratios measured in the averages of all mass spectra in each group and t-tests of the intensity of each individual peak. The targeted peptide ion signals are subsequently identified using MALDI-MS/MS in quadrupole-TOF and tandem-TOF instruments. This study found not only the proteins targeted and identified by a previous protein profiling experiment, but also detected additional proteins. These initial results are consistent with the known biology of pancreatic cancer or pancreatitis, but are not specific to those diseases.

Influences of Blood Sample Processing on Low–Molecular-Weight Proteome Identified by Surface-Enhanced Laser Desorption/Ionization Mass Spectrometry

“…Methods under development involve extraction/fractionation before tryptic digestion and high-resolution MALDI profiling of the peptides produced (48 ), as well as profiling of existing peptides with high-resolution MALDI after prefractionation based on reversed-phase magnetic particles and with subsequent data modeling (32 ). The latter study rigorously examined the circumstances necessary for generation of reproducible and complex spectra, including laser conditions and matrix composition.…”

Biomarker Characterization by MALDI–TOF/MS

“…In addition, other proteins with masses similar to that of biomarker proteins may give ion signals that overlap with each other on MALDI-TOF mass spectra. An alternative method for improving the characterization efficiency of low abundant or large intact proteins is through proteolytic digestion of the sample [304][305][306][307][308][309][310][311][312][313][314][315][316][317][318][319]. After digestion, the protein fragments detected by MALDI-TOF/MS in reflective mode within the lower mass range can be used to provide a characteristic high-resolution peptide profile.…”

“…After digestion, the protein fragments detected by MALDI-TOF/MS in reflective mode within the lower mass range can be used to provide a characteristic high-resolution peptide profile. If necessary, specific ions can be chosen for further MS/MS analysis for peptide sequencing [304][305][306][307][308][309][310][311][312][313][314][315][316][317][318][319]. Compared to intact protein profiling, the obtaining of highly resolved ion signals of representative peptides provide more sensitive and accurate diagnosis of proteins and extend the accessible protein molecular weight range.…”

The Use of Salivary as a Biometric Tool to Determine the Presence of Carcinoma of the Breast Among Women

“…There are numerous methods that are based on isotopically labeled protein modifying reagents to label or tag proteins to determine relative or absolute concentrations in complex mixtures. The higher resolution offered by the tandem Qq-TOF mass spectrometer is ideally suited to isotopically labeled applications (Gu et al, 2004;Koomen et al 2004;Ward et al, 1990). Applied Biosystems recently introduced iTRAQ reagents (Gu et al, 2004;Koomen et al 2004;Ward et al, 1990), which are amino reactive compounds that are used to label peptides in a total protein digest of a fluid such as saliva.…”

“…The higher resolution offered by the tandem Qq-TOF mass spectrometer is ideally suited to isotopically labeled applications (Gu et al, 2004;Koomen et al 2004;Ward et al, 1990). Applied Biosystems recently introduced iTRAQ reagents (Gu et al, 2004;Koomen et al 2004;Ward et al, 1990), which are amino reactive compounds that are used to label peptides in a total protein digest of a fluid such as saliva. The real advantage is that the tag remains intact through TOF-MS analysis; however, it is revealed during collision induced dissociation by MSMS analysis.…”