Acclaim pepmap 100 trapping column

Manufactured by Thermo Fisher Scientific

The Acclaim PepMap 100 trapping column is a chromatographic tool used for desalting and concentrating peptide samples prior to analysis. It features a 100 Å pore size and a C18 stationary phase, providing efficient separation and enrichment of peptides from complex mixtures.

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Acclaim PepMap 100 20mm × 0.075mm trapping column Acclaim PepMap 100 column Acclaim PepMap

4 protocols using acclaim pepmap 100 trapping column

Proteome Profiling of Cell Lysates

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SDS-lysed patient and cell line samples were processed and digested according to the filter-aided sample preparation (FASP) method [23 (link),24 (link)]. All of the filter-processed samples used 20 μg of protein material. Peptides from both patient and cell line samples were cleaned up with the Oasis HLB μElution (Waters, Milford, MA, USA) protocol.4.4. Liquid Chromatography (LC)-MS Analysis.
Dried peptides were dissolved in 20 μL of 2% acetonitrile (ACN) and 0.5% formic acid (FA). Differently preserved THP-1 and Molm-13 samples were analyzed on an Orbitrap Elite mass spectrometer equipped with a nanospray Flex ion source coupled to an Ultimate 3000 Rapid Separation LC system (both from Thermo Scientific, Waltham, MA, USA). Approximately 0.5 μg peptides were pre-concentrated and separated, as previously described [5 (link)]. Patient samples without or with PBS wash(es) were analyzed on a Q Exactive HF Orbitrap mass spectrometer equipped with an Easy-Spray (Thermo Scientific) coupled to an Ultimate 3000 Rapid Separation LC system. Approximately 0.6 μg peptides were pre-concentrated on a 2 cm × 75 µm ID Acclaim PepMap 100 trapping column and separated on a 50 cm × 75 µm ID Easy-Spray PepMap RSLC analytical column (both from Thermo Scientific). Bound peptides were eluted within a 195 min run using a binary gradient with buffer A (0.1% FA in water) and buffer B (0.1% FA in ACN).

Wangen R., Aasebø E., Trentani A., Døskeland S.O., Bruserud Ø., Selheim F, & Hernandez-Valladares M. (2018). Preservation Method and Phosphate Buffered Saline Washing Affect the Acute Myeloid Leukemia Proteome. International Journal of Molecular Sciences, 19(1), 296.

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Proteomic Analysis of A. alternata

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Proteomic analysis was performed in the Proteomics Unit of the Complutense University of Madrid, a member of ProteoRed, and supported by grant PT17/0019 of PE I+D+i 2013–2016, funded by ISCIII and ERDF.
Peptides digest from the A. alternata extract were analyzed by RP-LC-ESI-MS/MS in an EASY-nLC 1000 System coupled to the Q-Exactive HF mass spectrometer through a Nano-Easy spray source (all from Thermo Scientific).
Then, 1 µg of peptides was loaded first onto a pre-column Acclaim PepMap 100 Trapping column (Thermo Scientific, 20 mm × 75 μm ID, 3 μm C18 resin with 100 Å pore size) and then separated and eluted on an analytical reverse-phase Easy Spray column (Pepmap RSLC C18n 500 mm × 75 μm ID, 2 μm C18 resin with 100 Å pore size) with an integrated spray tip. A 150 min gradient of 2% to 35% Buffer B (100% acetonitrile, 0.1% formic acid) in Buffer A (0.1% formic acid) at a constant flow rate of 250 nl/min was used for the elution of peptides.
Data acquisition was performed with a Q-Exactive HF using data-dependent acquisition (DDA) and in positive mode with Xcalibur 4.0 software. From each Full MS (350–1800 Da) scan, the top 15 most abundant precursors, with charges of 2–6 in MS 1 scans, were selected for higher-energy collisional dissociation (HCD) fragmentation with a dynamic exclusion of 20 s.

Rodríguez D., Tabar A.I., Castillo M., Martínez-Gomariz M., Dobski I.C, & Palacios R. (2021). Changes in the Sensitization Pattern to Alternaria alternata Allergens in Patients Treated with Alt a 1 Immunotherapy. Journal of Fungi, 7(11), 974.

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eEF1A2 Protein Digestion and Peptide Analysis

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An overnight in-gel digestion of eEF1A2 protein was carried out under reducing and alkylating conditions (10 mM dithiothreitol, 55 mM iodoacetamide) with 12.5 ng/μL of bovine trypsin (Roche, 20 mM ammonium bicarbonate pH 8.5, 37 °C). The resulting peptides were freeze-dried in SpeedVac and resuspended in buffer A (2% acetonitrile, 0.1% formic acid). The analysis was performed using an EASY-nLC 1000 System coupled to the Q-Exactive HF mass spectrometer (1.8 Kv ion spray voltage and 250 ºC transfer temperature) through the Nano-Easy spray source. Separation was achieved by means of an Acclaim PepMap 100 Trapping column (Thermo Scientific) and an NTCC analytical column (100 Å; 3 μm; 150x0.75 mm; Nikkyo Technos Co. Ltd.). A 5-35% gradient of buffer B (100% acetonitrile, 0.1% formic acid) in buffer A was performed over 80 min, followed by a 30-45% gradient over 10 min at a constant flow rate of 250 nL/min. Both full-MS data-dependent acquisition (300-2800 Da m/z mass range, 100 ms scan time and a normalized collision energy of 27%) and semitarget parallel reaction monitoring (isolation of target peptide with a 2 m/z window from the inclusion list according to different charged states of the PTMs) were employed in positive mode using Xcalibur 4.0 software. Injection times of 200 ms for MS/MS were used at a mass resolution of 45000 Da.

Carriles A.A., Mills A., Muñoz-Alonso M.J., Gutiérrez D., Domínguez J.M., Hermoso J.A, & Gago F. (2021). Structural Cues for Understanding eEF1A2 Moonlighting. Chembiochem : a European journal of chemical biology, 22(2).

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Glycopeptide Analysis by LC-MS/MS

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The samples prepared were dissolved in 20 μL of 0.1% formic acid, and a 1 μL aliquot of the sample was injected into an EASY-nLC 1000 (Thermo Scientific) with an Acclaim PepMap 100 trapping column (75 μm×2 cm, nanoViper; Thermo Scientific) and a Nano HPLC Capillary Column (75 μm×120 mm, 3 μm, C18; Nikkyo Technos). The mobile phases were 0.1% formic acid (A buffer) and 0.1% formic acid in acetonitrile (B buffer). The glycopeptides were eluted at a flow rate of 0.3 μL/min with a linear gradient from 0 to 45% B over 55 min. Mass spectra were acquired on a Q Exactive mass spectrometer (Thermo Scientific) equipped with Nanospray Flex Ion Source (Thermo Scientific). The electrospray voltage was 2.0 kV, and the resolution was 70,000. Full mass scans were performed at a range of m/z 700–2,000, and product ion spectra were acquired data-dependently in the positive ion mode. The deglycosylated peptides were analyzed in a similar manner with the exception of full mass scans at m/z 350–2000.

Fujihira H., Masahara-Negishi Y., Tamura M., Huang C., Harada Y., Wakana S., Takakura D., Kawasaki N., Taniguchi N., Kondoh G., Yamashita T., Funakoshi Y, & Suzuki T. (2017). Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene. PLoS Genetics, 13(4), e1006696.

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