Coomassie brilliant blue (CBB)-stained portions of the gel containing protein spot of interest were excised, destained, and subjected to repeated dehydration rehydration steps prior to overnight digestion at 37°C with sequencing grade modified trypsin Gold (Promega, Madison, WI) as previously described (Kondo et al., 2006 (link); Miyachi et al., 2017 (link)). After digestion, tryptic peptides were extracted with 45% acetonitrile/0.1% trifluoroacetic acid and concentrated with a vacuum evaporator (Speed-Vac; Thermo Electron, San Jose, CA). The resulting peptide mixture was separated by reverse-phase chromatography (Tempo TM nano-LC system; Applied Biosystems, Foster City, CA) using a Pep Map C18 column. The eluting peptides were ionized by electrospray ionization and analyzed by QSTAR XL system (Applied Biosystems) for peptide mass finger-printing (PMF). Nanospray ionization was carried out by using an ion spray voltage at 900 V. The progress of each run was monitored by recording the total ion current for positive ions as a function of time in the m/z range of 400–1600 for MS and 140–1600 for MS/MS. The spectra were acquired in an information-dependent manner utilizing the Analyst QS 2.0 software. The other parameters set were interface temperature, 50°C; curtain gas flow, 1.13 L/min; focusing potential, 280 V; declustering potential 2, 15 V.
Qstar xl system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The QSTAR XL system is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform designed for analytical laboratories. It provides accurate mass measurements and effective separation of complex samples. The QSTAR XL system is a versatile analytical tool suitable for a wide range of applications.
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4 protocols using qstar xl system
1
Mass Spectrometry-Based Protein Identification
2
Serum Protein Profiling via iTRAQ-LC-MS/MS
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The iTRAQ-labeled serum samples were pooled and desalted on a C18 column (Sep-Pak vac, 1 cc, 50 mg, Waters, Germany). The desalted mixtures were diluted in buffer A (5% acetonitrile, 94.5% water, 0.1% trifluoroacetic acid) and fractionated on a ZORBAX 300SB-C18 Column (5μm, 300A, 0.5×23mm, Waters, Germany). connected to a 20AD HPLC system (Shimadzu, Kyoto, Japan). After chromatographic separation for 70 min, the sample was eluted by applying a linear gradient of buffer B (95% acetonitrile, 4.99% water, 0.1% trifluoroacetic acid) from 5% to 35% over 53 min. Fractions were collected every 2 min, giving a total of 35 fractions.
Fractions were dried in a rotary vacuum concentrator and dissolved in buffer C (5% acetonitrile, 0.1% trifluoroacetic acid). Peptides were separated on a ZORBAX 300SB-C18 Column (0.1 3 15 mm, 5 mm, 300 Å; Microm, Auburn, CA) by applying a linear gradient of buffer D (95% acetonitrile, 4.99% water, 0.1% trifluoroacetic) from 5% to 35% over 70 min. Eluted peptides were analyzed using a QSTAR XL system (Applied Biosystems Inc., USA) connected to a 20AD HPLC system (Shimadzu, Kyoto, Japan). Eluted peptides were scanned over the range of 400–1,800 m/z, and then over the range of 100–2,000 m/z. The pathway of iTRAQ-LC-MS/MS analysis is shown in Figure10 .
Fractions were dried in a rotary vacuum concentrator and dissolved in buffer C (5% acetonitrile, 0.1% trifluoroacetic acid). Peptides were separated on a ZORBAX 300SB-C18 Column (0.1 3 15 mm, 5 mm, 300 Å; Microm, Auburn, CA) by applying a linear gradient of buffer D (95% acetonitrile, 4.99% water, 0.1% trifluoroacetic) from 5% to 35% over 70 min. Eluted peptides were analyzed using a QSTAR XL system (Applied Biosystems Inc., USA) connected to a 20AD HPLC system (Shimadzu, Kyoto, Japan). Eluted peptides were scanned over the range of 400–1,800 m/z, and then over the range of 100–2,000 m/z. The pathway of iTRAQ-LC-MS/MS analysis is shown in Figure
3
Multi-dimensional Peptide Fractionation and Mass Spectrometry
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The combined peptide mixtures were fractionated via strong cation exchange (SCX) chromatography on a 20AD high-performance liquid chromatography (HPLC) system (Shimadzu; Kyoto, Japan) using a polysulfoethyl column (2.1 × 100 mm, 5 µm, 200 Å, Poly LC, Columbia, MD). Peptides were eluted with a linear gradient of 0–500 mM KCl (10 mM KH2PO4 in 25 % v/v acetonitrile, pH 2.6) for 60 min at a flow rate of 200 µL/min. In total, twenty fractions were collected.
Each fraction was dried, dissolved in 0.1 % FA (formic acid) aqueous solution, and analyzed on a QSTAR XL system (Applied Biosystems, China) interfaced with a 20AD HPLC system (Shimadzu, Kyoto, Japan). Peptides were separated on a reverse-phase Zorbax 300SB-C18 column (75 × 150 mm, 3 µm, 100 Å, Microm, Auburn, CA). The mobile phase was composed of 0.5 % formic acid in water (A) and acetonitrile (B). The flow rate was 400 nL/min with a gradient from 5 % to 45 % B over 70 min and 90 % B over 10 min. MS data were acquired automatically using Analyst QS 1.0 software Service Pack 8 (ABI/MDS SCIEX, Concord, Canada). Survey scans were acquired from 400 to 1800, with up to 4 precursors selected for MS/MS from m/z 100 to 2000. Curtain gas was set at 10, nitrogen was used as the collision gas, and the ionization tip voltage was 4000 V.
Each fraction was dried, dissolved in 0.1 % FA (formic acid) aqueous solution, and analyzed on a QSTAR XL system (Applied Biosystems, China) interfaced with a 20AD HPLC system (Shimadzu, Kyoto, Japan). Peptides were separated on a reverse-phase Zorbax 300SB-C18 column (75 × 150 mm, 3 µm, 100 Å, Microm, Auburn, CA). The mobile phase was composed of 0.5 % formic acid in water (A) and acetonitrile (B). The flow rate was 400 nL/min with a gradient from 5 % to 45 % B over 70 min and 90 % B over 10 min. MS data were acquired automatically using Analyst QS 1.0 software Service Pack 8 (ABI/MDS SCIEX, Concord, Canada). Survey scans were acquired from 400 to 1800, with up to 4 precursors selected for MS/MS from m/z 100 to 2000. Curtain gas was set at 10, nitrogen was used as the collision gas, and the ionization tip voltage was 4000 V.
4
HPLC Fractionation and Mass Spectrometry Analysis
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Chromatographic separation of the pooled samples was performed on a 20AD high-performance liquid chromatography (HPLC) system (Shimadzu; Kyoto, Japan). Tryptically digested and labeled peptides were first fractionated using a strong cation exchange liquid chromatograph on a 2.1 mm × 150 mm, 3.5 µm, 300 Å column (Waters Corporation, Milford, MA). The sample was loaded onto the column and eluted stepwise by injecting salt plugs of ten molar concentrations (25, 50, 75, 100, 150, 200, 300, 400, 500, and 1,000 mM NH4Ac). Ten fractions were collected from the strong cation exchange column. Each fraction was then loaded across a ZORBAX 300SB-C18 RP column (5 µm, 300 Å, 0.1 × 150 mm; Michrom BioResources, Auburn, CA) and analyzed on a QSTAR XL System (Applied Biosystems) coupled with a 20AD HPLC system (Shimadzu). The flow rate used for separation on the reversed-phase (RP) column was 0.4 ml/min. Buffer A consisted of 5% acetonitrile, 95% water, and 0.1% formic acid; Buffer B consisted of 95% acetonitrile, 5% water, and 0.1% formic acid. Elution was performed using a gradient ranging from 5% to 45% of Buffer B over 90 min [34 (link)].
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