Abstract
It is a major challenge to develop effective sequence database search algorithms to translate molecular weight and fragment mass information obtained from tandem mass spectrometry into high quality peptide and protein assignments. We investigated the peptide identification performance of Mascot and X!Tandem for mass tolerance settings common for low and high accuracy mass spectrometry. We demonstrated that sensitivity and specificity of peptide identification can vary substantially for different mass tolerance settings, but this effect was more significant for Mascot. We present an adjusted Mascot threshold, which allows the user to freely select the best trade-off between sensitivity and specificity. The adjusted Mascot threshold was compared with the default Mascot and X!Tandem scoring thresholds and shown to be more sensitive at the same false discovery rates for both low and high accuracy mass spectrometry data.
Highlights
It is a major challenge to develop effective sequence database search algorithms to translate molecular weight and fragment mass information obtained from tandem mass spectrometry into high quality peptide and protein assignments
Performance of the Mascot Identity Threshold—Mass errorcorrected spectra were submitted to Mascot and searched at 2-Da, 1-Da, 100-ppm, 50-ppm, 20-ppm, and 5-ppm maximum mass deviation (MMD) settings, whereas all other parameters were fixed
Under more stringent settings (5 ppm) the Mascot identity threshold (MIT) median decreased to 24, whereas the interquartile range increased to 2. These results suggest that the MIT adapts with changing search space and performs more like a global cutoff based on the narrow variation in thresholds
Summary
Sample 1—A nuclear protein extract of murine embryonic stem cells (2 mg/ml) was reduced with 2 mM dithiothreitol (Sigma) at 70 °C for 10 min followed by alkylation with 20 mM iodoacetamide (Sigma) at room temperature for 30 min. 10 g of total protein was separated on a NuPAGE Novex 4 –12% Bis-Tris polyacrylamide gel (Invitrogen). The entire gel lane was excised into 48 bands, destained with 50% acetonitrile, and subsequently digested with sequencing grade trypsin (Roche Applied Science) overnight. Peptides were extracted with 5% formic acid, 50% acetonitrile twice and vacuum-dried in a SpeedVac (Thermo Fisher Scientific). Sample 2—A standard protein set of 48 human proteins (Sigma, Universal Proteomics Standard Set UPS1) was reduced with Tris(2carboxyethyl)phosphine hydrochloride (TCEP), alkylated with iodoacetamide as above, followed by digestion in solution with sequencing grade trypsin (Roche Applied Science) overnight. Peptides were analyzed with on-line nano-LC-MS/MS on an LTQ FT (Thermo Fisher Scientific), a hybrid linear ion trap and a 7-tesla Fourier transform ion cyclotron resonance mass spectrometer, coupled with an Ultimate 3000Nano/Capillary LC System (Dionex). The instrument was externally calibrated using the standard calibration mixture of caffeine (peptide sequence MRFA), and Ultramark 1600
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