Pierce hela protein digest standard

Manufactured by Thermo Fisher Scientific

Sourced in Germany

Pierce HeLa Protein Digest Standard is a reference material for the identification and quantification of proteins. It is a complex mixture of peptides derived from the digestion of HeLa cell proteins, providing a representative sample of the human proteome. This standard can be used to validate and optimize protein identification and quantification workflows.

Automatically generated - may contain errors

Lab products found in correlation

Ms grade trypsin, by Promega (4 mentions) Lys c, by Promega (3 mentions) Dithiothreitol (dtt), by Thermo Fisher Scientific (3 mentions) Iodoacetamide iaa, by Thermo Fisher Scientific (3 mentions) Irt kit, by Biognosys (2 mentions) Chromasolv lc ms water, by Honeywell (2 mentions) Acetonitrile, by Honeywell (2 mentions) Mass spec compatible yeast extract, by Promega (2 mentions) Xbridge premier peptide beh c18, by Waters Corporation (1 mentions) Orbitrap eclipse mass spectrometer, by Thermo Fisher Scientific (1 mentions) Ultimate 3000 lc system, by Thermo Fisher Scientific (1 mentions) 9 mm short thread tpx vial withintegrated glass micro insert, by Thermo Fisher Scientific (1 mentions) Lc ms grade water, by Honeywell (1 mentions) Mass spec compatible yeast digest, by Promega (1 mentions) Massprep e coli digest standard, by Waters Corporation (1 mentions) Optima lc ms grade, by Thermo Fisher Scientific (1 mentions) N dodecyl β d maltoside ddm, by Merck Group (1 mentions) Angiotensin 1, by Merck Group (1 mentions) Lc ms grade formic acid, by Thermo Fisher Scientific (1 mentions) Phosphoric acid solution, by Merck Group (1 mentions)

18 protocols using pierce hela protein digest standard

LC-MS/MS Analysis of HeLa Digest

Check if the same lab product or an alternative is used in the 5 most similar protocols

Final LC–MS/MS
analysis for method evaluation was performed using an Orbitrap Eclipse
mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled to
an UltiMate 3000 LC system (Thermo Fisher Scientific, Germering, Germany).
For these short gradients (5, 10, and 15 min), a self-packed trap
column (XBridge Premier Peptide BEH C18, 2.5 μm, 150 μm
i.d., 3 cm length, 130 Å; Waters, Milford, MA, USA) and a self-packed
analytical column (XBridge Premier Peptide BEH C18, 2.5 μm,
75 μm i.d., 5 cm length, 130 Å; Waters, Milford, MA, USA)
were used. The mobile phases were as follows: for the (i) positive
ion mode, (A) 0.1% FA in H₂O and (B) 0.1% FA and 95% ACN
in water and for the (ii) negative ion mode, (A) 2.5 mM imidazole
and 3% IPA in water and (B) 2.5 mM imidazole, 3% IPA, and 95% ACN
in water. The loading solvent was in both cases mobile phase A. The
gradient was from 2 to 40% B in the corresponding minutes of the gradient
(5, 10, or 15 min) at a flow rate of 1.5 μL/min. Full MS scans
were acquired with 240,000 resolution. For the Thermo Scientific Pierce
HeLa protein digest standard, 500 ng of the sample was injected. In
the case of the digestion with the different enzymes, 1 μg of
HeLa digest was loaded on the column. For each condition (positive
and negative), each chromatogram time (5, 10, and 15 min), and each
enzyme (trypsin, GluC, LysC, and AspN), the sample was injected in
quadruplicate.

Penanes P.A., Gorshkov V., Ivanov M.V., Gorshkov M.V, & Kjeldsen F. (2023). Potential of Negative-Ion-Mode Proteomics: An MS1-Only Approach. Journal of Proteome Research, 22(8), 2734-2742.

+ Open protocol

+ Expand

Cross-linking Mass Spectrometry Protocol

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Pierce HeLa Protein Digest Standard
(Thermo Fisher Scientific) was used for MS parameter optimization
as well as for final column benchmarking measurements. 20 μg
of peptide pellets were dissolved in LC/MS grade water with 0.1% (v/v)
trifluoroacetyl (TFA) and diluted to the required peptide concentration
in autosampler vials (Fisherbrand 9 mm Short Thread TPX Vial with
integrated Glass Micro-Insert; Cat. no. 11515924). All liquid handling
was done as fast as possible without unnecessary time gaps, with the
aim to minimize sample losses on plastics and glass surfaces.
For the cross-linking experiment, synthetic peptides generated by
Beveridge and coworkers were cross-linked using disuccinimidyl dibutyric
urea (DSBU), as described in their paper.^{23 (link)} The final cross-linked peptide mix was merged either with an equal
amount of tryptic HeLa peptides (Pierce HeLa Protein Digest Standard
dissolved in 0.1% TFA) to obtain a 1:1 spiked system or with 5 times
the amount of tryptic HeLa peptides to obtain a 1:5 spiked system.
A total amount of 1 μg (either using the cross-linked peptide
mix only or total peptide after spiking) of the peptide was used for
each LC–MS/MS analysis.

Stejskal K., Jeff O.D., Matzinger M., Dürnberger G., Boychenko A., Jacobs P, & Mechtler K. (2022). Deep Proteome Profiling with Reduced Carryover Using Superficially Porous Microfabricated nanoLC Columns. Analytical Chemistry, 94(46), 15930-15938.

+ Open protocol

+ Expand

Benchmarking of MS Standards for QC

Check if the same lab product or an alternative is used in the 5 most similar protocols

QC1 samples tested in Thermo and Sciex instruments (LTQ-Orbitrap Velos Pro, LTQ-Orbitrap XL, LTQ-q-Orbitrap Fusion Lumos; and QqQ 5500 QTRAP) corresponded to 1 vial of 500 pmol of commercially available “Trypsin-digested BSA MS Standard (CAM modified)” from New England Biolabs with part number P8108S. The 500 pmol of dried digested bovine serum albumin (BSA) were dissolved with 500 μL of 0.1% formic acid in water and then 15 μL were diluted with 285 μL of 0.1% formic acid in water to 50 fmol/μL. A total of 0.5 μL were injected in each analysis, which corresponded to 25 fmol of BSA.
QC2 samples tested in Thermo and Sciex instruments corresponded to Pierce HeLa protein digest standard from Thermo Fisher Scientific (Part number: 88329). The commercial product is a vial of 20 μg dried digested HeLa extract which are dissolved in 200 μL of 0.1% formic acid in water to a final concentration of 100 ng/μL. A total amount of 1 μL (100ng) is injected per analysis.
All quality control samples used in this work were commercially available.

Chiva C., Olivella R., Borràs E., Espadas G., Pastor O., Solé A, & Sabidó E. (2018). QCloud: A cloud-based quality control system for mass spectrometry-based proteomics laboratories. PLoS ONE, 13(1), e0189209.

+ Open protocol

+ Expand

HeLa Peptide Digest Standard Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

The QC standard was a commercially available peptide digest derived from the HeLa human cervical cancer cell line, the Pierce HeLa Protein Digest Standard (Thermo Fisher Scientific), resuspended at a concentration of 1 µg µL⁻¹ in HPLC-grade water containing 0.1% (v/v) formic acid and supplemented with eleven non-naturally occurring synthetic peptides from the iRT kit (Biognosys) at a ratio 1:30 v/v. The detailed preparation instructions are described in Supplementary Note 1.

Xuan Y., Bateman N.W., Gallien S., Goetze S., Zhou Y., Navarro P., Hu M., Parikh N., Hood B.L., Conrads K.A., Loosse C., Kitata R.B., Piersma S.R., Chiasserini D., Zhu H., Hou G., Tahir M., Macklin A., Khoo A., Sun X., Crossett B., Sickmann A., Chen Y.J., Jimenez C.R., Zhou H., Liu S., Larsen M.R., Kislinger T., Chen Z., Parker B.L., Cordwell S.J., Wollscheid B, & Conrads T.P. (2020). Standardization and harmonization of distributed multi-center proteotype analysis supporting precision medicine studies. Nature Communications, 11, 5248.

+ Open protocol

+ Expand

Mass Spectrometry Sample Preparation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dithiothreitol (DTT), iodoacetamide (IAA), Pierce formic acid (LC-MS grade), and Pierce HeLa protein digest standard were purchased from Thermo Fisher Scientific. CHROMASOLV™ LC-MS water and acetonitrile were products of Honeywell (Charlotte, NC, USA), MS-grade trypsin and Lys-C were from Promega (Madison, WI, USA). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.

Cong Y., Motamedchaboki K., Misal S.A., Liang Y., Guise A.J., Truong T., Huguet R., Plowey E.D., Zhu Y., Lopez-Ferrer D, & Kelly R.T. (2020). Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell. Chemical Science, 12(3), 1001-1006.

+ Open protocol

+ Expand

Single-cell proteomics workflow for cell lines

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Pierce HeLa Protein Digest Standard (Thermo Fisher, Waltham, MA) was diluted in LC–MS grade water (Honeywell, Charlotte, NC) to provide single-cell-sized (200 pg) aliquots for system characterization. For MBR library creation, 10 ng aliquots of the same HeLa digest and of a K562 peptide digest (Promega) were prepared. HeLa, Jurkat, and K562 cell lines were purchased from ATCC (Manassas, VA). These were cultured at 37 °C in 5% CO₂, with 10% fetal bovine serum and 1% penicillin-streptomycin added to Dulbecco’s modified Eagle’s media (VWR, Radnor, PA) for HeLa cells and to Gibco RPMI 1640 media 1× (Life Technologies, NY) for Jurkat and K562 cells. Cells were passaged every 2 days or upon reaching 70% confluency. The three cell lines were harvested and washed three times with phosphate-buffered saline (PBS) and centrifuged to remove debris and supernatant at 200g for 5 min after each wash. The three cell lines were then counted and resuspended to a concentration of approximately 10⁴ cells/mL, after which single cells were isolated into nanowells using the CellenONE X1 platform (Cellenion, Lyon, France) using gating parameters similar to those described previously.^{13 (link)} The cells were then prepared using the nanoPOTS workflow.^{10 ,29 (link)}

Webber K.G., Truong T., Johnston S.M., Zapata S.E., Liang Y., Davis J.M., Buttars A.D., Smith F.B., Jones H.E., Mahoney A.C., Carson R.H., Nwosu A.J., Heninger J.L., Liyu A.V., Nordin G.P., Zhu Y, & Kelly R.T. (2022). Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform. Analytical chemistry, 94(15), 6017-6025.

+ Open protocol

+ Expand

Preparation of Controlled Protein Digest Samples

Check if the same lab product or an alternative is used in the 5 most similar protocols

The controlled samples A and B were prepared from the Pierce HeLa Protein Digest Standard (Thermo Fisher Scientific), the Mass Spec-Compatible Yeast Digest (Promega), and the MassPREP E. coli Digest Standard (Waters). Each digest was resuspended at a concentration of 1 µg µL⁻¹ with HPLC-grade water containing 0.1% (v/v) formic acid. Sample A was prepared by mixing human, yeast, and E. coli protein digests at 65%, 15%, and 20% w/w, respectively. Sample B was prepared by mixing human, yeast, and E. coli protein digests at 65%, 30%, and 5% w/w, respectively. The iRT kit (Biognosys) was added to each of the controlled samples at a ratio 1:30 v/v. For analysis, 2 µl of each sample was injected. The detailed preparation instructions are described in Supplementary Note 1.

+ Open protocol

+ Expand

LC-MS Proteomics Sample Preparation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dithiothreitol (DTT) and iodoacetamide (IAA) were products of Thermo Fisher Scientific (Waltham, MA) and were freshly prepared in 50 mM ammonium bicarbonate buffer before use. Water with 0.1% formic acid (v/v, Optima LC/MS grade), which served as mobile phase A, acetonitrile with 0.1% formic acid (v/v, Optima LC/MS grade), which served as mobile phase B, and Pierce HeLa protein digest standard were also from Thermo Fisher. MS-grade trypsin and Lys-C were from Promega (Madison, WI). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.

Cong Y., Liang Y., Motamedchaboki K., Huguet R., Truong T., Zhao R., Shen Y., Lopez-Ferrer D., Zhu Y, & Kelly R.T. (2020). Improved Single-Cell Proteome Coverage Using Narrow-Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry. Analytical chemistry, 92(3), 2665-2671.

+ Open protocol

+ Expand

Peptide Quantification via LC-UV

Check if the same lab product or an alternative is used in the 5 most similar protocols

Final peptide amounts were determined by separating an aliquot of each sample on a liquid chromatography-UV system equipped with a monolith column based. The final amounts were determined through comparison to a peak area of 100 ng of Pierce HeLa protein digest standard (PN 88329; ThermoFisher Scientific). Peptide solution was frozen at −70 °C before further processing.

Monteil V., Kwon H., John L., Salata C., Jonsson G., Vorrink S.U., Appelberg S., Youhanna S., Dyczynski M., Leopoldi A., Leeb N., Volz J., Hagelkruys A., Kellner M.J., Devignot S., Michlits G., Foong-Sobis M., Weber F., Lauschke V.M., Horn M., Feldmann H., Elling U., Penninger J.M, & Mirazimi A. (2023). Identification of CCZ1 as an essential lysosomal trafficking regulator in Marburg and Ebola virus infections. Nature Communications, 14, 6785.

+ Open protocol

+ Expand

Mass Spectrometry Protein Digestion

Check if the same lab product or an alternative is used in the 5 most similar protocols

Pierce HeLa Protein Digest Standard was purchased from ThermoFisher Scientific (Waltham, MA). N-Dodecyl-β-D-maltoside (DDM) and Mastoparan peptide (CAS 72093–21-1, a cell-permeable synthetic peptide with a sequence identical to Vespula lewisii), were purchased from Sigma-Aldrich (St. Louis, MO). Dithiothreitol (DTT) and iodoacetamide (IAA) were purchased from ThermoFisher Scientific and were freshly prepared in 50 mM ammonium bicarbonate buffer before use. MS-grade trypsin was purchased from Promega (Madison, WI). All aqueous solutions were prepared using 18.2 MΩ deionized water produced from a Barnstead Nanopure Infinity system (Los Angeles, CA). Unless otherwise noted, all other unmentioned reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Dou M., Tsai C.F., Piehowski P.D., Wang Y., Fillmore T.L., Zhao R., Moore R.J., Zhang P., Qian W.J., Smith R.D., Liu T., Kelly R.T., Shi T, & Zhu Y. (2019). Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples. Analytical chemistry, 91(15), 9707-9715.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!