In a separate set of experiments, glycopeptide enrichments of sIL-15Rα peptide mixtures of the clinical-grade cGMP lot of hetIL-15 were performed using hydrophilic interaction liquid chromatography (HILIC) in a micro-column solid phase extraction (SPE) format prior to LC-MS/MS analysis. The dried peptide mixtures were redissolved in 10 μl of HILIC loading solvent consisting of 80 % (vol/vol) ACN in 0.1 % (vol/vol) TFA (aq). The enrichments were carried out as described previously with minor modifications [26 (link), 27 (link)]. In short, a stationary phase consisting of washed ZIC-HILIC resin (particle size, 10 μm; pore size, 200 Å) (Sequant/Merck) was used to pack a HILIC SPE column (approximately length, 10 mm) in a GeLoader tip (Eppendorf). The column was equilibrated in 20 μl loading solvent and the peptide mixtures were loaded manually at low flow. The column was washed twice with 10 μl loading solvent before the retained glycopeptides were eluted with 10 μl eluting solvent consisting of 0.1 % (vol/vol) TFA (aq). The eluate was dried and taken up in 20 μl 0.1 % (vol/vol) formic acid (aq) and used for multiple LC-MS/MS injections.
Geloader tip
Manufactured by Eppendorf
Sourced in Germany, United States
The GELoader tip is a disposable, single-use pipette tip designed for use with Eppendorf pipettes. It is intended for general laboratory applications involving the transfer of liquids.
Automatically generated - may contain errors
Lab products found in correlation
Sequencing grade trypsin solution, by Promega (2 mentions)
Sequencing grade trypsin, by Promega (2 mentions)
Poros 50 r2 rp column material, by Thermo Fisher Scientific (2 mentions)
Bovine fetuin, by Merck Group (2 mentions)
Milli q system, by Merck Group (2 mentions)
Glycolic acid, by Merck Group (2 mentions)
Pngase f, by New England Biolabs (2 mentions)
Maldi plate, by Thermo Fisher Scientific (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Mineral oil, by Nacalai Tesque (1 mentions)
Nuclease free water, by Qiagen (1 mentions)
Trypsin, by Agilent Technologies (1 mentions)
Turbosequest software, by Thermo Fisher Scientific (1 mentions)
Agilent 1100 series lc system, by Agilent Technologies (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Sequencing grade modified trypsin, by Merck Group (1 mentions)
Lysc protease, by Merck Group (1 mentions)
N buoh, by Merck Group (1 mentions)
O dichlorobenzene, by Merck Group (1 mentions)
28 protocols using geloader tip
1
Glycopeptide Enrichment and LC-MS/MS Analysis
2
Purification of AMC-derivatized Metabolites
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Prior to purification of the AMC-derivatized metabolites from HPCs, 10 µl of the internal standard was added to each sample. Prior to addition to the samples, the internal standard was purified as described in the following: a TiO2 micro-column was packed in a 20 µl GELoader tip (Eppendorf). A small plug of a C8 filter was put at the constricted end of the tip to hold back the TiO2 beads. The 5 µm Titansphere TiO2 beads (GL Science, Japan) were suspended in 0.6% acetic acid in 80% acetonitrile and loaded into the micro-column. The long tip below the C8 plug was cut away to reduce the backpressure of the column. The total HPC sample was loaded onto the micro-column and was washed twice with 30 µl of 0.6% acetic acid in 80% acetonitrile. Subsequently, the sample was washed with 30 µl water and was eluted first with 30 µl 5% ammonia and second with 3 µl 0.6% acetic acid in 80% acetonitrile. The elution was collected directly in a low binding glass vial containing 45 µl 10% trifluoroacetic acid to acidify the eluate. Finally, the sample was concentrated in a vacuum centrifuge.
3
In-Gel Trypsin Digestion for Mass Spectrometry
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The procedure for in-gel digestion of protein spots from Coomassie Blue stained gels was carried out as described in [9 (link)]. In brief, protein spots were excised from stained gels and cut into pieces. The gel pieces were washed for 1 h at room temperature in 25 mM ammonium bicarbonate buffer, pH 7.8, containing 50% (v/v) acetonitrile (ACN) and dehydrated in a SpeedVac for 10 min and rehydrated in 10 μL (20 ng/μL) of sequencing grade trypsin solution (Promega, WI). After incubation in 25 mM ammonium bicarbonate buffer, pH 7.8, at 37°C overnight, the tryptic peptides were extracted with 5 μL of 0.5% TFA containing 50% (v/v) ACN for 40 min with mild sonication. The extracted solution was reduced to 1 μL in a vacuum centrifuge. The resulting peptide solution was desalted using a reversed-phase column [10 (link)] and subjected to mass spectrometric analysis. A GEloader tip (Eppendorf, Hamburg, Germany) constricted was packed with Poros 20 R2 resin (PerSpective Biosystems, MA). After an equilibration with 10 μL of 5% (v/v) formic acid, the peptide solution was loaded on the column and washed with 10 μL of 5% (v/v) formic acid. The bound peptides were eluted with 1 μL of α-cyano-4-hydroxycinnamic acid (CHCA) (5 mg/mL in 50% (v/v) ACN/5% (v/v) formic acid) and dropped onto a MALDI plate (96 × 2; Applied Biosystems, CA).
4
Peptide Desalting and Concentration for MS
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Custom-made chromatographic columns were used for desalting and concentrating the peptide mixture prior to MS analysis. A column consisting of 100–300 nL of Poros reverse phase R2 material (20–30 μm bead size, Perseptive Biosystems, Framingham, MA, USA) was packed in a constricted GELoader tip (Eppendorf, Hamburg, Germany). A 10 mL syringe was used to force liquid through the column by applying gentle air pressure. Thirty microliters of the peptide mixture from the digestion supernatant was diluted in 30 μL of 5% formic acid, loaded onto the column, and washed with 30 μL of 5% formic acid. For MS/MS analyses, the peptides were eluted with 1.5 μL of 50% methanol/49% H2O/1% formic acid directly into a pre-coated borosilicate nano-electrospray needle (New Objective, Woburn, MA, USA).
5
Yeast Cell Preparation for HR-MACS Analysis
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The sample preparation for HR-MACS was performed under a stereomicroscope. The S. cervisiae cells (ca. 300 × 106 cells in 4 μl of D2O) were pipetted into a 550/400-μm capillary sealed at one end (with epoxy) using a micropipette equipped with a 20 μl GELoader® tip (Eppendorf, US). Very gentle centrifugation was applied for 2–5 s to remove air bubbles and ensure the cells displaced into the solenoid region. Within the coil detection region there are ca. 19 × 106 cells. The top of the sample capillary was then sealed with hot paraffin wax to prevent leakage during sample spinning, and inserted into the HR-MACS resonator already fitted inside the MAS rotor. The entire sample-preparation procedure was restricted to no more than 2 min to minimize the chances of sample tampering and degradation.
6
Peptide Desalting and Concentration for MS/MS
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The custom-made chromatographic columns were used for desalting and concentration of the peptide mixture. A column consisting of 100–300 nL of Poros reverse phase R2 material (PerSeptive Biosystems, Framingham, MA, USA) was packed in a constricted GELoader tip (Eppendorf, Hamburg, Germany). The liquid was forced gently into the column using a 10-mL syringe. Thirty microliters of the peptide mixture from the digestion supernatant was diluted with 30 μL of 5 % formic acid, loaded onto the column, and washed with 30 μL of 5 % formic acid. Peptides were eluted with 1.5 μL of 50 % methanol/49 % H2O/1 % formic acid directly into a precoated borosilicate nanoelectrospray needle (New Objective, Woburn, MA, USA) for analysis by tandem mass spectrometry (MS/MS).
7
Peptide Purification for Mass Spectrometry
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Custom-made chromatographic columns were used for desalting and concentrating the peptide mixture prior to mass spectrometric analysis. A column consisting of 100–300 nL of Poros Reversed-phase R2 material (20–30-μm beads, PerSeptive Biosystems, Framingham, MA, USA) was packed in a constricted GELoader Tip (Eppendorf, Hamburg, Germany). A 10-mL syringe was used to force liquid through the column by applying gentle air pressure. Thirty microliters of the peptide mixture from the digestion supernatant was diluted with 30 μL of 5% formic acid, loaded onto the column, and washed with 30 μL of 5% formic acid. For analyses by tandem mass spectrometry (MS/MS) analyses, peptides were eluted with 1.5 μL of 50% methanol/49% H2O/1% formic acid directly into a precoated borosilicate nanoelectrospray needle (EconoTip™, New Objective, Woburn, MA, USA).
8
Desalting Peptide Mixtures for MS/MS
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Custom-made chromatographic columns were utilized for the desalting of the peptide mixture prior to mass spectrometric analysis. The column consisted of 100–300 nL of Poros reverse-phase R2 material (bead size of 20–30 μm, PerSeptive Biosystems) within a constricted GELoader tip (Eppendorf, Hamburg, Germany). Gentle air pressure was applied via a 10 mL syringe to facilitate liquid flow through the column. Subsequently, 30 μL of the peptide mixture from the digested supernatant was diluted in 30 μL of 5% formic acid, loaded onto the column, and washed with an additional 30 μL of 5% formic acid. For tandem mass spectrometry (MS/MS) analysis, the peptides were eluted using 1.5 μL of a solution containing 50% methanol, 49% H2O, and 1% formic acid.
9
Peptide Purification and MS Analysis
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Custom-made chromatographic columns were used for desalting and concentration of the peptide mixture before mass spectrometric analysis. A column consisting of 100–300 nL of Poros reverse-phase R2 material (20–30 μm bead size, PerSeptive Biosystems) was packed in a constricted GELoader tip (Eppendorf, Hamburg, Germany). A 10-mL syringe was used to force the liquid through the column by applying gentle air pressure. Next, 30 μL of the peptide mixture from the digested supernatant was diluted in 30 μL of 5% formic acid, loaded onto the column, and washed with 30 μL of 5% formic acid. For analyses by tandem mass spectrometry (MS/MS), the peptides were eluted with 1.5 μL of 50% methanol/49% H2O/1% formic acid.
10
Glycopeptide Enrichment Using PAM-Lactose@SiO2
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Glycopeptide enrichment was performed in a micro-scale SPE mode. Firstly, an Eppendorf GELoader tip was packed with 1 mg PAM-g-lactose0.11@SiO2 material after being slurried with acetonitrile (ACN) to obtain the SPE micro-column. Then the tip was conditioned and equilibrated with 20 μL 50%ACN/20 mM ammonium formate (NH4FA) (pH 3.8) and 20 μL 80%ACN/20 mM NH4FA (pH 3.8), respectively. When tryptic digests dissolved in 80%ACN/20 mM NH4FA was loaded, the micro-column was washed twice with 20 μL 75% ACN/20 mM NH4FA (pH 3.8) and 20 μL 75%ACN/0.1%formic acid (FA), respectively. Subsequently, the captured glycopeptides were eluted with 30 μL 50%ACN/5 mM NH4HCO3 (pH 8.6). Similar enrichment procedure was also employed for reference material PAM@SiO2. As for commercial material control experiments, TiO255 (link) and Sepharose60 (link) were used to enrich SGs, performed according to the reported method with minor modification.
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