Workflow for analysis of high mass accuracy salivary data set using MaxQuant and ProteinPilot search algorithm

Abstract

LTQ Orbitrap data analyzed with ProteinPilot can be further improved by MaxQuant raw data processing, which utilizes precursor-level high mass accuracy data for peak processing and MGF creation. In particular, ProteinPilot results from MaxQuant-processed peaklists for Orbitrap data sets resulted in improved spectral utilization due to an improved peaklist quality with higher precision and high precursor mass accuracy (HPMA). The output and postsearch analysis tools of both workflows were utilized for previously unexplored features of a three-dimensional fractionated and hexapeptide library (ProteoMiner) treated whole saliva data set comprising 200 fractions. ProteinPilot's ability to simultaneously predict multiple modifications showed an advantage from ProteoMiner treatment for modified peptide identification. We demonstrate that complementary approaches in the analysis pipeline provide comprehensive results for the whole saliva data set acquired on an LTQ Orbitrap. Overall our results establish a workflow for improved protein identification from high mass accuracy data.

Figure 1

Overview of data set and workflow. (A) Data set for comparison of effect of HPMA in a data set using ProteinPilot. (B) Workflow for comparison of effect of HPMA in a data set using ProteinPilot.

Figure 2

ProteinPilot descriptive statistics for a salivary data set. (A) Mass Accuracy plots from ProteinPilot searches of MaxQuant processed and ReAdW-processed peaklists. The distribution of the frequency of spectra identified by ProteinPilot has been plotted against precursor Delta ppm. (B) Distributions of peptide and protein scores of confident identifications from ProteinPilot searches. The distributions of the frequency of spectra identified by ProteinPilot at 5% local FDR was plotted against peptide score (Sc). (C) Numbers of protein identifications from ProteinPilot for small human salivary data set (data set 1 in Figure 1B) at 5% local FDR. MGF files were created with ReAdW or MaxQuant. Similar observations were made for distinct peptides and spectral level data set (See Supporting Information Fig S1).

Figure 3

Combined MaxQuant and ProteinPilot workflow and results from saliva proteome analysis. (A) Combined workflow of MaxQuant and ProteinPilot software. (B) Features of salivary data set studied using MaxQuant-processed files and Mascot/ProteinPilot.