MALDI-TOF mass spectrometry analysis was performed using the Voyager DE-PRO (Applied Biosystems, Foster City, CA, USA) mass spectrometer as previously described (34 (link)). Briefly, silver stained band was in gel-destained with K3[Fe(CN)6] and Na2S2O3, reduced with dithiothreitol, S-alkylated with iodoacetamide, and subsequently digested with trypsin. The tryptic peptide extracts were desalted by μZip-TipC18 (Millipore) and loaded on the MALDI target, using the dried droplet technique and α-cyano-4-hydroxycinnamic acid as matrix. The resulting mass spectrum, was elaborated using the DataExplorer software (Applied Biosystems) and manually inspected to obtain the corresponding peak lists. Internal mass calibration was done using trypsin autolysis fragments at m/z 842.5100, 1045.5642, and 2211.1046 Da. Peptide mass fingerprinting was compared to the theoretical masses from the Swiss-Prot.
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Lab products found in correlation
4800 maldi tof tof mass spectrometer, by Thermo Fisher Scientific (4 mentions)
4000 series explorer software, by Thermo Fisher Scientific (2 mentions)
Voyager de pro, by Thermo Fisher Scientific (1 mentions)
μzip tipc18, by Merck Group (1 mentions)
Mono q 5 50 gl, by GE Healthcare (1 mentions)
Voyager de str maldi tof mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ammonium bicarbonate, by Promega (1 mentions)
4800 maldi tof tof analyzer, by Thermo Fisher Scientific (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Voyager de str mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Voyager de str biospectrometry workstation, by Thermo Fisher Scientific (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Gps explorer software, by Thermo Fisher Scientific (1 mentions)
Anti he4 antibody, by Santa Cruz Biotechnology (1 mentions)
Mascot algorithm, by Matrix Science (1 mentions)
4000 series explorer, by Thermo Fisher Scientific (1 mentions)
4800 plus maldi tof tof analyser, by AB Sciex (1 mentions)
5800 maldi tof tof analyzer, by AB Sciex (1 mentions)
Empore c8 disk, by 3M (1 mentions)
Mascot distiller, by Matrix Science (1 mentions)
24 protocols using data explorer software
1
MALDI-TOF Mass Spectrometry Protocol
2
Protein Identification and Domain Analysis
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The identity of full-length recombinant lymphostatin was confirmed by in-gel protein digest and peptide analysis. Excised gel-bands were incubated at a porcine trypsin:lymphostatin ratio of ∼1:30, in 50 mm ammonium bicarbonate overnight at 32 °C (Promega). Peptides were identified by matrix-assisted laser desorption ionization (MALDI) mass spectroscopy on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) using an α-cyano-4-hydroxycinnamic acid matrix. The spectral data were processed using Data Explorer software (Applied Biosystems) and the MASCOT NCBInr database searched against the peptide mass map (Matrix Science). To investigate the domain structure of lymphostatin, purified protein was incubated with trypsin at a ratio of 375:1, at 21 °C, to give limited digestion. Aliquots were removed at 1, 2, 3, and 4 h and the reaction stopped by boiling samples adjusted with 2 mm EDTA and 2 mm PMSF in SDS-PAGE loading buffer. Digest products were separated by SDS-PAGE and individual bands were subjected to in-gel tryptic digestion and MALDI-TOF mass spectroscopy as described above. Peptide masses were compared with the sequence of full-length rLifA using GPMAW 9.2 software, mass tolerance 50 ppm (19 (link)). Fragment F1 was purified to homogeneity from other digest products by ion-exchange chromatography (Mono-Q 5/50 GL; GE Healthcare) as described above.
3
Phosphopeptide Analysis by MALDI-TOF/TOF
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Phosphopeptide from in vitro kinase assay was performed by 4800 MALDI TOF/TOF Analyzer (Applied Biosystems, Foster City, CA, USA). 0.5 μL of enriched phosphopeptides was mixed with 0.5 μL of matrix (20 mg/mL 2,5-dihydroxybenzoic acid (DHB) in 50% ACN and 1% H3PO4). MS was performed by positive reflector mode with the setting of 20 kV accelerated voltage, 16% grid voltage, and low-mass gate of 1000 Da. One spectrum was composed by 1200 laser pulses. Data-Explorer software (Applied Biosystems) was used for raw spectra processing of baseline subtraction and noise removement.
4
Protein Identification in Xanthomonas
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All data were processed using the Data Explorer Software (Applied Biosystems, CA). Proteins were identified by correlation of tandem mass spectra and Xac genome data bank available at NCBI, using the MASCOT™ software (Matrix Science, version 2.1). One missed cleavage per peptide was allowed and an initial mass tolerance of 0.05 Da was used in all searches. Cysteines were assumed to be carbamidomethylated, and variable modification of methionine (oxidation) was allowed. To evaluate the false positive rate of this approach, a reversed sequence databank (a database in which the sequences have been reversed) containing the same number of proteins as in the Xac database was constructed. Identification was considered positive if it matched at least one unique peptide.
5
MALDI-TOF-MS Protein Identification Protocol
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The aforementioned pooled extracts were dried in a lyophilizer and the extracts were redissolved in 1 µl extraction buffer (50 µl acetonitrile, 20 µl trifluoroacetic acid and 930 µl distilled water) and 1 µl matrix solution (α-acyano-4-hydroxycinnamic acid) and targeted onto a MALDI-TOF plate. Following drying the samples completely onto the targeting plate, MALDI-TOF-MS was performed using a Voyager-DE STR mass spectrometer (Applied Biosystems; Thermo Fisher Scientific, Inc.) equipped with delay ion extraction. Mass spectra were acquired over a mass range between 800 and 3,000 Da. The peptide mass peak list was processed using DataExplorer software (version 4.8; Applied Biosystems; Thermo Fisher Scientific, Inc.) to search the protein against the SwissProt database (www.ebi.ac.uk/uniprot ) using the Mascot-Peptide Mass Fingerprint program (www.matrixscience.com ). The following parameters were used for database searches: Taxonomy, Homo sapiens (human); cleavage specificity, trypsin with one missed cleavage allowed; peptide tolerance of 100 p.p.m. for the fragment ions; and allowed modifications, cysteine carbamidomethyl (fixed) and oxidation of methionine (variable). The MOWSE scores (>56) and species were considered to identify the correct protein from the Mascot results list.
6
MALDI-TOF MS Analysis of Peptides and Proteins
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Peptides and glycopeptides were dissolved in water and mixed with the corresponding matrix solution (1:1 v/v) and 1 µL of the mixture was applied to the MALDI target and allowed to dry at room temperature. For analysis of synthetic peptides and conjugates, DHB (10 mg/mL) in ACN:water:TFA (70:30:0.1 v/v/v) was chosen as matrix. For peptide digests, saturated CHCA in ACN:water:TFA (70:30:0.1 v/v/v) was used. For protein analysis, SA (10 mg/mL) in ACN:water:TFA (70:30:0.1 v/v/v) was used.
MS spectra were recorded on a Voyager-DE STR Biospectrometry workstation (Applied Biosystems, Foster City, CA, USA) equipped with a N2 laser (337 nm). Peptides and glycopeptides were measured in reflectron mode and positive polarity, except for Sia-containing probes, which were measured in both positive and negative modes. Proteins were measured in the linear mode and positive polarity. External calibration was performed using the SequazymeTM Peptide Mass Standard Kit (PerSeptive Biosystems, Framingham, MA, USA) of the desired range. Data were processed with the Data Explorer Software (Applied Biosystems).
MS spectra were recorded on a Voyager-DE STR Biospectrometry workstation (Applied Biosystems, Foster City, CA, USA) equipped with a N2 laser (337 nm). Peptides and glycopeptides were measured in reflectron mode and positive polarity, except for Sia-containing probes, which were measured in both positive and negative modes. Proteins were measured in the linear mode and positive polarity. External calibration was performed using the SequazymeTM Peptide Mass Standard Kit (PerSeptive Biosystems, Framingham, MA, USA) of the desired range. Data were processed with the Data Explorer Software (Applied Biosystems).
7
HE4 Protein Identification by IP-MALDI-TOF
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OVCAR-3 cell was immunoprecipitated using an anti-HE4 antibody (Santa Cruz, goat, CA) and combined with 30 ul of protein A/G PLUS agarose (Santa Cruz) by rotating for 1 h at 4°C. The eluents were loaded onto SDS-PAGE gel and Coomassie brilliant blue-stained. The expressed bands were excised and processed for in-gel trypsin digestion and subjected to MALDI-TOF-MS analysis. anti-HE4 antibody was replaced by goat IgG (Bioss, China) for negative control. The peptide and proteins were identified from the MS/MS spectra using the MASCOT algorithm (Matrix Science, Boston, MA). Peptide mass fingerprinting was carried out using the MASCOT search engine from GPS Explorer software (Applied Biosystems, Foster City, CA). Mass spectra used for manual denovo sequencing were annotated with the Data Explorer soft-ware (Applied Biosystems).
8
MALDI-TOF MS Analysis of Glycopeptides
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MALDI-TOF (matrix-assisted laser desorption/ionisation-time of flight) MS was carried out on all glycopeptides used in this study. HPLC peak fractions were diluted 1:1 with 3,4-diaminobenzophenone dissolved at 10 mg/mL in 75% (v/v) acetonitrile or with 2,5-dihydroxybenzoic acid dissolved at 20 mg/mL in 70% (v/v) methanol. The glycopeptide/matrix mix (1 µL) was then spotted on a 384-well target plate (AB Sciex) and left to dry at room temperature overnight. The following day samples were analysed in positive or negative-ion mode using a 4800 Plus MALDI–TOF/TOF Analyser (AB Sciex) with 4000 Series Explorer (Applied Biosystems). Tandem MS of glycopeptides was carried out using collision-induced dissociation fragmentation. Data were extracted with the Data Explorer software (Applied Biosystems), and semi-manual data analysis was carried out with the open-source software Skyline (MacCoss Lab). For final graphic editing, CorelDraw software was used.
9
MALDI-TOF/TOF Mass Spectrometry Protocol
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Mass spectra were acquired in negative or positive reflector mode on Applied Biosystems/MDS SCIEX MALDI 4800 Plus TOF/TOF spectrometer. Analyte ionization was achieved with a 355 nm Nd:YAG laser firing at 200 Hz rate. Laser fluence was within the 6000–6900 AU range. The analyzer was operated in delayed extraction mode from 250 to 650 ns. Typically, 1000 laser shots were accumulated. The raw spectrum was analyzed and edited (Gaussian smoothing, filter width: 99 points) using Data Explorer software, Applied Biosystems.
10
Glycomic Analysis of N-glycans
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The MS and MS/MS data were processed employing Data Explorer Software from Applied Biosystems. The processed spectra were annotated using a glycobioinformatics tool, GlycoWorkBench [15 (link)]. Based on known biosynthetic pathways and susceptibility to PNGase F digestion, all N-glycans are presumed to have a Manα1–6(Manα1–3)Manβ1–4GlcNAcβ1–4GlcNAc core structure [16 (link),17 (link)]. The symbolic nomenclature used in the spectra annotation is the same as the one used by the Consortium for Functional Glycomics (CFG) (http://www.functionalglycomics.org/ ) and the Essentials for Glycobiology on-line textbook (http://www.ncbi.nlm.nih.gov/books/NBK1931/figure/ch1.f5/?report=objectonl ). Reproducibility and comparability were assessed by ANOVA (see Figure S5 ).
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