Protein tyrosine-O-sulfation analysis by exhaustive product ion scanning with minimum collision offset in a NanoESI Q-TOF tandem mass spectrometer.
ABSTRACT Tyrosine-O-sulfated peptides were studied by nanoESI Q-TOF mass spectrometry and were found to exhibit an abundant loss of SO3 in positive ion mode under the usually nonfragmenting conditions of survey spectrum acquisition. A new strategy for the detection of tyrosine-O-sulfated peptides in total protein digests was designed based on exhaustive product ion scanning at the collision offset conditions typical for the recording of survey spectra (minimum collision offset). From these data, Q-TOF neutral loss scans for loss of 80/z and Q-TOF precursor ions scans were extracted. The specificity of this approach for analysis of tyrosine-O-sulfation was tested using a tryptic digest of bovine serum albumin spiked with sulfated hirudin (1:1 and 1000:1 molar ratio of BSA to sulfated hirudin, respectively) and using an in-solution digest of the recombinant extracellular domain of thyroid stimulating hormone receptor (ECD-TSHr). For both examples, the combination of in silico neutral loss scans for 80/z and subsequent in silico precursor ion scans resulted in a specific identification of sulfated peptides. In the analysis of recombinant ECD-TSHr, a doubly sulfated peptide could be identified in this way. Surprisingly, approximately 1/4 of the product ion spectra acquired from the tryptic digest of ECD-TSHr at minimum collision offset exhibited sequence-specific ions suitable for peptide identification. Complementary ion pairs were frequently observed, which either were b2/y(max-2) pairs or were induced by cleavage N-terminal to proline. MS/MS analysis at minimum collision offset followed by extraction of neutral loss and precursor ion scans is ideally suited for highly sensitive detection of analyte ions which exhibit facile gas-phase decomposition reactions.
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ABSTRACT: Recombinant factor IX (rFIX) is increasingly used to treat patients with hemophilia B. CHO (Chinese Hamster Ovary) cells are commonly used for the production of rFIX but they have a limited capacity for introducing post-translational modifications (PTM) leading to incomplete γ-carboxylation, low phosphorylation and sulfation profiles as compared with plasma-derived preparations. Imperfect PTM might have an impact on the activity of Factor IX molecule. Several studies in animal models as well as clinical trials have previously reported a lower recovery of rFIX compared to plasma-derived FIX concentrates. In the present study, we aimed to produce a rFIX having a profile of PTM similar to plasma-derived FIX, using human hepatoma cell line HuH-7. We showed that rFIX produced by HuH-7 cells followed the classical intracellular pathway before secretion. In addition, improved PTM were associated with fully active molecule compared to plasma-derived and recombinant control FIX molecules. Secreted rFIX presented as a single band at the correct molecular weight. HuH-7 cellular clones were obtained and they secreted a biologically active human FIX. FIX was then purified for a detailed evaluation of PTM. Glycosylation and sialylation profiles were similar to plasma-derived and rFIX and mass spectrometry analysis demonstrated the presence of phosphorylated and sulfated forms of rFIX. These data strongly support that HuH-7 cells may represent an effective cellular system for production of rFIX exhibiting PTM similar to plasma-derived FIX.Thrombosis Research 09/2012; 130(5):e266-73. · 3.13 Impact Factor
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ABSTRACT: Unambiguous differentiation between isobaric sulfated and phosphorylated tyrosine residues (sTyr and pTyr) of proteins by mass spectrometry is challenging, even using high resolution mass spectrometers. Here we show that upon negative ion mode collision-induced dissociation (CID), pTyr- and sTyr-containing peptides exhibit entirely different modification-specific fragmentation patterns leading to a rapid discrimination between the isobaric covalent modifications using the tandem mass spectral data. This study reveals that the ratio between the relative abundances of [M-H-80](-) and [M-H-98](-) fragment ions in ion-trap CID and higher energy collision dissociation (HCD) spectra of singly deprotonated +80 Da Tyr-peptides can be used as a reliable indication of the Tyr modification group nature. For multiply deprotonated +80 Da Tyr-peptides, CID spectra of sTyr- and pTyr-containing sequences can be readily distinguished based on the presence/absence of the [M-nH-79]((n-1)-) and [M-nH-79-NL]((n-1)-) (n=2, 3) fragment ions (NL=neutral loss).Journal of the American Society for Mass Spectrometry 09/2011; 22(12):2256-68. · 3.59 Impact Factor
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ABSTRACT: This review describes some of the more interesting and imaginative ways in which mass spectrometry has been utilized to study a number of important post-translational modifications over the past two decades; from circa 1990 to 2013. A diverse range of modifications is covered, including citrullination, sulfation, hydroxylation and sumoylation. A summary of the biological role of each modification described, along with some brief mechanistic detail, is also included. Emphasis has been placed on strategies specifically aimed at detecting target modifications, as opposed to more serendipitous modification discovery approaches, which rely upon straightforward product ion scanning methods. The authors have intentionally excluded from this review both phosphorylation and glycosylation since these major modifications have been extensively reviewed elsewhere. © 2014 Wiley Periodicals, Inc. Mass Spec RevMass Spectrometry Reviews 03/2014; · 7.74 Impact Factor