Interpreting raw biological mass spectra using isotopic mass‐to‐charge ratio and envelope fingerprinting

Abstract

RATIONALE: Soft ionization, high-resolution mass spectrometry is widely used to characterize large biological molecules, such as proteins. Deconvolution ('deisotoping') of isotopic envelopes (iEs) in biological mass spectra into monoisotopic or average masses is challenging due to low signals and heavily overlapped iEs, resulting in many wrong interpretations. METHODS: Isotopic envelopes (iEs) are directly used without deisotoping to identify biological molecules. An algorithm, isotopic mass-to-charge ratio (m/z) and envelope fingerprinting (iMEF), was implemented in the ProteinGoggle search engine for top-down intact protein database searching. iMEF combines isotopic m/z fingerprinting (iMF) and isotopic envelop fingerprinting (iEF), where 'Isotopic mass-to-charge ratio' means the m/z value of the most abundant isotopic peak within the iE of a precursor or product ion. iMF is used to 'fish' precursor or product ion candidates from the database, which is pre-built and contains all iE information (precursor and product ions) of all proteoforms of the studied system. iEF identifies matching precursor or product ions. A protein is finally identified with user-specified total number of matching product ions and post-translational modification scores. RESULTS: The working principles of iMEF and ProteinGoggle, and the definition of a set of related parameters and scoring metrics, are illustrated with high-resolution tandem mass spectrometric analysis of a mixture of ubiquitin and the HUMAN histone H4 proteoforms. Ubiquitin was confidently identified from its CID, ETD, and HCD spectra with 57, 91, and 66 matching product ions, respectively; 125 proteoforms were confidently found from the H4 dataset. The locations of PTMs in 54 and 6 isoforms were partially and fully identified. CONCLUSIONS: Database search with iMEF bypasses 'deisotoping' avoiding associated errors, and also provides full quality control of matching precursor and product ions and finally protein IDs. Overlapped iEs of different product ions could also be confidently unwrapped in situ. Improvement and addition of more functionalities and utilities of ProteinGoggle are underway.

Mass measurement accuracy of the Orbitrap in intact proteome analysis

“…The analysis includes three replicates each day across six continuous days. The obtained raw datasets were searched using ProteinGoggle 2.0 based on the iMEF (isotopic mass‐to‐charge ratio and envelope fingerprinting) algorithm …”

Site‐ and Structure‐Specific Quantitative N‐Glycoproteomics Using RPLC‐pentaHILIC Separation and the Intact N‐Glycopeptide Search Engine GPSeeker

“…To leverage well‐known N‐glycosylation microheterogeneity where multiple isomeric structures sharing the same monosaccharide composition on the same N‐glycosite may have different expression and pathological relevance, we recently developed a GPSeeker‐centered structure‐specific isotopic‐labeling quantitative N‐glycoproteomics pipeline (Xiao & Tian, ). The intact N‐glycopeptide search engine GPSeeker adopts the search algorithm of isotopic mass‐to‐charge ratio and envelope fingerprinting (iMEF; Li & Tian, ; Xiao et al., ), which has been successfully used for database searches of intact proteins (Xiao, Yu, & Tian, ) and N‐glycans (Xiao, Han, & Tian, ; Xiao, Wang, Shen, Han, & Tian, ). With isotopic dimethyl labeling, structure‐specific RPLC‐pentaHILIC 2DLC separation, and a GPSeeker database search, differentially expressed intact N‐glycopeptides (DEGPs) in HCC HepG2 cells relative to normal liver LO2 cells have been successfully characterized both qualitatively with comprehensive structures and quantitatively with accurate relative abundance ratios.…”

Top‐down characterization of mouse core histones

“…Here, we report our top‐down characterization of mouse core histones extracted from fibroblast cells NIH/3T3. With nanoRPLC‐MS/MS analysis and database search by ProteinGoggle 2.0, 547 protein species with combinatorial PTMs were identified with spectrum‐level FDR ≤ 1%; PTMs in 51 protein species were unambiguously localized with site‐determining product ions. Only 16 protein species were identified in the previously mentioned literature focusing on top‐down proteomics of histones from mouse.…”