Dataset Information

JPST000068 monolith HILIC-HeLa [Reanalysis: JPST000068]

ABSTRACT: A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 Î¼m for nucleosides and a permeability of 2.1 Ã— 10âˆ’13 m2, which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies. [Original project description]

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: RPXD034323 | JPOST Repository | Sat Apr 01 00:00:00 BST 2023

REPOSITORIES: jPOST

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			Action	DRS
	131228tk-HILIC-monolith-4h-1ug-1.pilotFin.txt	Txt
	131228tk-HILIC-monolith-4h-1ug-1.txt	Txt
	131228tk-HILIC-monolith-4h-1ug-2.pilotFin.txt	Txt
	131228tk-HILIC-monolith-4h-1ug-2.txt	Txt
	131228tk-HILIC-monolith-4h-1ug-3.pilotFin.txt	Txt

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Project description:Recent advances in mass spectrometric (MS) instruments resulting in high mass resolution and high duty cycles have allowed of very deep coverage of proteomes. However, even with state of the art, high duty cycle instruments, the number of proteins identified with a conventional single liquid chromatography-tandem MS (LC/MS/MS) analysis is typically limited and fractionating protein samples prior to LC/MS/MS analysis is crucial for increasing both the analytical dynamic range and proteome coverage. In order to achieve near comprehensive identification of proteomes two-dimensional (2D) chromatography has been an invaluable tool. This first dimension is typically performed with µL/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range while the second dimension is typically performed with nL/min flow rates and capillary columns with small inner diameters. Typically, reverse phase liquid chromatography (RPLC) provides high peak resolution of peptides and achieves higher peak capacities than strong cation exchange chromatography (SCX) due to the faster chromatographic partitioning. Another advantage of RPLC is that the mobile phases used generate much cleaner samples for downstream analyses, while the incorporation of a desalting step for SCX in order to eliminated the high salt concentrations that can significantly decrease the analytical sensitivity of the MS analysis due to ionization problems. When operated at widely different pH values (e.g., 1st dimension pH 10 and 2nd dimension pH 3) 2D RPLC-RPLC provides separation orthogonality comparable to that of the combination of SCX-RPLC. The difference of separation selectivity between the low and high pH (HpH) RPLC comes from the changes in the charge distribution within peptide chains upon altering pH of the eluent. Herein we describe a method in which tryptic peptides are separated in the first dimension by HpH-RPLC and fractions are collected every 90 s over an 80 min gradient. For the second dimension, each of these fractions is individually run by the standard low pH RPLC method. The implementation of the HpH pre-fractionation allows for short second dimension gradients for bottom up LC/MS/MS and yields very deep (e.g. thousands of identifications) protein coverage at reasonable measurement time.

Project description:Proteome analyses of human induced pluripotent cells (iPSC) were carried out by liquid chromatography–tandem mass spectrometry systems using meter-scale monolithic silica-C18 capillary columns without pre-fractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast cell (FBC) lysates (4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithicsilica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. Consequently, 98,977 non-redundant tryptic peptides from 9,510 proteins (corresponding to 8,712 genes), including low abundant protein groups such as 329 protein kinases, were successfully identified from triplicate measurements within 10 days. The obtained proteome profiles of 8 cells were successfully categorized into two groups, iPSC and FBC, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially-modified protein abundance index approach combined with UniProt keyword enrichment analysis reveals that the iPSC group contains more ‘transcription regulation’ proteins and the FBS group contains more ‘transport’ related proteins. This simplified “one-shot” proteomics approach with long monolithic columns enables to accomplish the rapid, deep, sensitive and reproducible proteome analysis. Sample pretreatment was carried out according to the PTS protocol with some modifications shown below. Proteins were extracted from the pellets with 12 mM SDC, 12 mM SLS, and 50 mM ammonium bicarbonate, reduced with 10 mM dithiothreitol at room temperature for 30 min; and alkylated with 55 mM iodoacetamide in the dark at room temperature for 30 min. The protein mixture was 5-fold diluted with 50 mM ammonium bicarbonate and Lys-C/trypsin digestion was performed. An equal volume of ethyl acetate was added to the eluent solution, and the mixture was acidified with 0.5% trifluoroacetic acid (final concentration). The mixture was shaken for 1 min and centrifuged at 15,700 x g for 2 min, and then the aqueous phase was collected. Tryptic peptides were desalted with reversed phase-StageTips. Raw data files were searched against the IPI human database v3.87 (91,464 sequences) using AB SCIEX ProteinPilot v4.0 with the set parameter of “Instrument: TripleTOF 5600”and “Digestion: trypsin”. A precursor mass tolerance of 0.05 Da and a fragment ion mass tolerance of 0.1 Da were employed. Carbamidomethylation of cysteine, oxidation of methionine, phosphorylation of serine, threonine and tyrosine, deamidation of asparagine, glutamine, N-terminal pyro-glutamic acid of glutamine or glutamic acid and protein N-terminal acetylation were only accepted, and peptides were rejected if the peptide confidence was below 95%, the delta mass was over 0.05 Da, the charge state was more than 5 and the number of missed cleavage was more than 2. For protein identification, peptides were grouped into ‘protein group’ based on the rules previously established. Then, at least two confidently identified peptides per protein were used for protein identification. In addition, single peptides with higher confidence (p<0.01) were allowed for protein identification.

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JPST000068 monolith HILIC-HeLa [Reanalysis: JPST000068]

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