Adapting Data-Independent Acquisition for Mass Spectrometry-Based Protein Site-Specific N‑Glycosylation Analysis

A hallmark of protein N-glycosylation is extensive heterogeneity associated with each glycosylation site. In human cells, the constituent glycoforms differ mostly in numerous ways of extensions from an invariable trimannosyl core and terminal modifications. The efficient identification of these glycoforms at the glycopeptide level by mass spectrometry (MS) requires a precursor sampling technique that is not dictated by signal intensity or by preset targets during MS2 data acquisition. We show here that the recently developed data-independent acquisition (DIA) approach is best suited to this demanding task. It allows postacquisition extraction of glycopeptide-specific fragment-ion chromatograms to be aligned with that of precursor MS1 ion by nanoLC elution time. For any target glycoprotein, judicious selection of the most favorable MS1/MS2 transitions can first be determined from prior analysis of a purified surrogate standard that carries similar site-specific glycosylation but may differ in its exact range of glycoforms. Since the MS2 transitions to be used for extracting DIA data is common to that glycosylation site and not dictated by a specific MS1 value, our workflow applies equally well to the identification of both targeted and unexpected glycoforms. Using a case example, we show that, in targeted mode, it identified more site-specific glycoforms than the more commonly used data-dependent acquisition method when the amount of the target glycoprotein was limited in a sample of high complexity. In discovery mode, it allows detection, with supporting MS2 evidence, of under-sampled glycoforms and of those that failed to be identified by searching against a predefined glycan library owing to unanticipated modifications

Adapting Data-Independent Acquisition for Mass Spectrometry-Based Protein Site-Specific N‑Glycosylation Analysis

A hallmark of protein N-glycosylation is extensive heterogeneity associated with each glycosylation site. In human cells, the constituent glycoforms differ mostly in numerous ways of extensions from an invariable trimannosyl core and terminal modifications. The efficient identification of these glycoforms at the glycopeptide level by mass spectrometry (MS) requires a precursor sampling technique that is not dictated by signal intensity or by preset targets during MS2 data acquisition. We show here that the recently developed data-independent acquisition (DIA) approach is best suited to this demanding task. It allows postacquisition extraction of glycopeptide-specific fragment-ion chromatograms to be aligned with that of precursor MS1 ion by nanoLC elution time. For any target glycoprotein, judicious selection of the most favorable MS1/MS2 transitions can first be determined from prior analysis of a purified surrogate standard that carries similar site-specific glycosylation but may differ in its exact range of glycoforms. Since the MS2 transitions to be used for extracting DIA data is common to that glycosylation site and not dictated by a specific MS1 value, our workflow applies equally well to the identification of both targeted and unexpected glycoforms. Using a case example, we show that, in targeted mode, it identified more site-specific glycoforms than the more commonly used data-dependent acquisition method when the amount of the target glycoprotein was limited in a sample of high complexity. In discovery mode, it allows detection, with supporting MS2 evidence, of under-sampled glycoforms and of those that failed to be identified by searching against a predefined glycan library owing to unanticipated modifications

Adapting Data-Independent Acquisition for Mass Spectrometry-Based Protein Site-Specific N‑Glycosylation Analysis

A hallmark of protein N-glycosylation is extensive heterogeneity associated with each glycosylation site. In human cells, the constituent glycoforms differ mostly in numerous ways of extensions from an invariable trimannosyl core and terminal modifications. The efficient identification of these glycoforms at the glycopeptide level by mass spectrometry (MS) requires a precursor sampling technique that is not dictated by signal intensity or by preset targets during MS2 data acquisition. We show here that the recently developed data-independent acquisition (DIA) approach is best suited to this demanding task. It allows postacquisition extraction of glycopeptide-specific fragment-ion chromatograms to be aligned with that of precursor MS1 ion by nanoLC elution time. For any target glycoprotein, judicious selection of the most favorable MS1/MS2 transitions can first be determined from prior analysis of a purified surrogate standard that carries similar site-specific glycosylation but may differ in its exact range of glycoforms. Since the MS2 transitions to be used for extracting DIA data is common to that glycosylation site and not dictated by a specific MS1 value, our workflow applies equally well to the identification of both targeted and unexpected glycoforms. Using a case example, we show that, in targeted mode, it identified more site-specific glycoforms than the more commonly used data-dependent acquisition method when the amount of the target glycoprotein was limited in a sample of high complexity. In discovery mode, it allows detection, with supporting MS2 evidence, of under-sampled glycoforms and of those that failed to be identified by searching against a predefined glycan library owing to unanticipated modifications

Novel LC-MS² Product Dependent Parallel Data Acquisition Function and Data Analysis Workflow for Sequencing and Identification of Intact Glycopeptides

Data dependent acquisition (DDA) of higher collision energy dissociation (HCD)-MS² followed by electron transfer dissociation (ETD)-MS² upon detection of glycan-specific oxonium is one of the better approaches in current LC-MS² analysis of intact glycopeptides. Although impressive numbers of glycopeptide identification by a direct database search have been reported, false positives remained high and difficult to determine. Even in cases when the peptide backbones were correctly identified, the exact glycan moieties were often erroneously assigned. Any attempt to fit the best glycosyl composition match by mass only is problematic particularly when the correct monoisotopic precursor cannot be determined unambiguously. Taking advantage of a new trihybrid Orbitrap configuration, we experimented with adding in a parallel ion trap collision induced dissociation (CID)-MS² data acquisition to the original HCD-product dependent (pd)-ETD function. We demonstrated the feasibility and advantage of identifying the peptide core ion directly from edited HCD-MS² data as an easy way to reduce false positives without compromising much sensitivity in intact glycopeptide positive spectrum matches. Importantly, the additional CID-MS² data allows one to validate the glycan assignment and provides insight into possible glycan modifications. Moreover, it is a viable alternative to deduce the glycopeptide backbone particularly in cases when the peptide backbone cannot be identified by ETD/HCD. The novel HCD-pd-CID/ETD workflow combines the best possible decision tree dependent MS² data acquisition modes currently available for glycoproteomics within a rapid Top Speed DDA duty cycle. Additional informatics can conceivably be developed to mine and integrate the rich information contained within for simultaneous N- and O-glycopeptide analysis

Modifying an Insect Cell <i>N</i>‑Glycan Processing Pathway Using CRISPR-Cas Technology

Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect <i>N</i>-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the <i>N</i>-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the <i>fdl</i> gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man₃GlcNAc₂), and led to the production of partially elongated, mammalian-type complex <i>N</i>-glycans (GlcNAc₂Man₃GlcNAc₂) in S2 cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit insect cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing insect and mammalian <i>N</i>-glycan processing pathways. Finally, the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures

Characterization of DS-acutobin.

<p>(A) Analysis of DS-acutobin mass by ESI-MS spectrometry. (B) Comparison of human fibrinogen hydrolyses by 20 µg/ml of acutobin and DS-acutobin. The products were analyzed by SDS-PAGE after 1∼24 h. Abbreviations used: A, acutobin; D, DS-acutobin; ctl, control with fibrinogen only.</p

Coagulation of human plasma by acutobin and its various glycoforms.

<p>(A) Coagulation of human plasma was monitored spectrophotometrically at 37°C after addition of the indicated enzymes (final 0.25 µg/ml). The curves are averaged results of three experiments. (B) Coagulation times of human plasma in the presence of various amounts of the enzymes in a total volume of 150 µl were determined by a coagulation analyzer. Data shown are based on the averaged results of two or three experiments.</p

Thermal stability of different glycoforms of acutobin.

<p>The indicated enzymes were incubated at 37°C, 55°C and 65°C for 1 h. The hydrolytic activities toward Tosyl-Gly-Pro-Arg-<i>p</i>-nitroanilide were assayed at 25°C and an enzyme concentration of 2.5 µg/ml. The remaining activity of each enzyme after incubation at 37°C was taken as 100%.</p

Euglobulin-clot-lysis assay.

<p>The stabilities of clots formed by the indicated enzymes were compared based on the results of this assay. The curves are averaged results of two experiments.</p

Catalytic efficiencies of acutobin, DS-acutobin, ATBs, and PNG-enzymes toward chromogenic substrate Tosyl-Gly-Pro-Arg-<i>p</i>-nitroanilide.

<p>Catalytic efficiencies of acutobin, DS-acutobin, ATBs, and PNG-enzymes toward chromogenic substrate Tosyl-Gly-Pro-Arg-<i>p</i>-nitroanilide.</p