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Pinpoint software

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Pinpoint software is a bioinformatics tool developed by Thermo Fisher Scientific. It provides a platform for the analysis and interpretation of proteomics data.

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Lab products found in correlation

Zorbax rrhd eclipse plus c18 column, by Agilent Technologies (3 mentions) 6490 ifunnel triple quadrupole lc ms system, by Agilent Technologies (3 mentions) Tsq vantage, by Thermo Fisher Scientific (1 mentions) Pge2 d4, by Cayman Chemical (1 mentions) Pgf2a d4, by Cayman Chemical (1 mentions) Tsq vantage emr, by Thermo Fisher Scientific (1 mentions) Q exactive quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions) Ultimate 3000, by Thermo Fisher Scientific (1 mentions) Quan browser, by Thermo Fisher Scientific (1 mentions) Thermo excalibur 2, by Thermo Fisher Scientific (1 mentions)

11 protocols using pinpoint software

1

Nano-LC-MS/MS Peptide Separation

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The pre-column was connected in-line with an analytical column (50µm ID × 10cm packed with 5µm beads (YMC gel, ODS-AQ, 12nm, S-5 µm, AQ12S05)) with integrated electrospray emitter tip [Martin 2000]. A 140 minute gradient from aqueous (0.2M acetic acid) and organic phase (0.2M HOAc, 70% MeCN) was used to separate peptides with a flow rate of 200 nL/min and a flow split > 99% and a spray voltage of 1800 V. Isolation widths of 0.7 were used for both Q1 and Q3 and collected for 0.05 seconds. A width of 0.01m/z was collected around each transition for quantification. Collision energies were determined per sequence with Thermo Pinpoint software.
Curran T.G., Zhang Y., Ma D.J., Sarkaria J.N, & White F.M. (2015). MARQUIS: A Multiplex Method for Absolute Quantification of Peptides and Post-Translational Modifications. Nature communications, 6, 5924.
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2

Peptide Quantification Using Pinpoint

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Raw files obtained from PRM acquisitions were analyzed by Pinpoint software (version 1.4.0 Thermo Fisher Scientific). Ion chromatograms were extracted with a mass tolerance of 10 ppm using all b and y ions. The composite MS/MS spectrum of each targeted peptide was reconstructed from the area under curves (AUCs) of all the transitions (Fig. 4b, Additional file 3). The AUC for each modified peptide was deduced by addition of corresponding common co-eluted fragment ions AUCs observed in all replication and groups [14 (link)].
Jagadeeshaprasad M.G., Batkulwar K.B., Meshram N.N., Tiwari S., Korwar A.M., Unnikrishnan A.G, & Kulkarni M.J. (2016). Targeted quantification of N-1-(carboxymethyl) valine and N-1-(carboxyethyl) valine peptides of β-hemoglobin for better diagnostics in diabetes. Clinical Proteomics, 13, 7.
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3

Quantitative Mass Spectrometry Protocol

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Raw files recorded for each sample were analyzed using Pinpoint software (Thermo Fisher Scientific, USA) [53 (link)], and peptide XICs were extracted. Pinpoint was used for identification and visualization of transitions, as well as manual verification of co-elution of heavy and endogenous peptides. In the first SF set, in order to control for technical variation between the samples, XIC corresponding to each endogenous peptide replicate were divided by the XIC corresponding to the average of the two housekeeping proteins. This value was then averaged amongst the two replicate runs, to obtain “XIC Average normalized to housekeeping proteins”. In SF set II, in order to control for technical variation between the samples and obtain a more robust quantitative value for our proteins of interest, the XIC value corresponding to each endogenous peptide, was divided by the XIC value corresponding to each spiked-in heavy peptide, in order to obtain a L:H (light:heavy) ratio. Since we added a known amount of each heavy peptide to our samples prior to analysis, we used the L:H ratio to calculate the relative concentration of each endogenous peptide corresponding to our proteins of interest.
Cretu D., Prassas I., Saraon P., Batruch I., Gandhi R., Diamandis E.P, & Chandran V. (2014). Identification of psoriatic arthritis mediators in synovial fluid by quantitative mass spectrometry. Clinical Proteomics, 11(1), 27.
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4

Optimized Targeted Proteomics Workflow

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PeptideAtlas (http://www.peptideatlas.org/) was utilized to select the most commonly observed 3–4 tryptic peptides for the proteins of interest. Their presence was confirmed using our LC-MS/MS identification data. The uniqueness of peptides was verified using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi). To identify peptide fragments (transitions) to monitor, in silico peptide fragmentation was performed using Pinpoint software (Thermo Fisher Scientific, USA) and 5–6 transitions were selected for each peptide. For method optimization, digested pooled samples of SF used in our LC-MS/MS analysis, were loaded onto a C-18 column (Proxeon Biosystems, FL) coupled to a triple quadrupole mass spectrometer (TSQ Vantage; Thermo Fisher Scientific, USA), and approximately 350 transitions were monitored in 6 subsequent runs. Three transitions of the most intense peptides were used for subsequent quantification assays (Table 2).
Cretu D., Prassas I., Saraon P., Batruch I., Gandhi R., Diamandis E.P, & Chandran V. (2014). Identification of psoriatic arthritis mediators in synovial fluid by quantitative mass spectrometry. Clinical Proteomics, 11(1), 27.
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5

Proteotypic Peptide Screening and Quantification

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Proteins with higher proteotypic peptide response were screened as specific candidate proteins by in silico digestion using Pinpoint software (version 1.0 Thermo Fisher ScientiFIc). MS/MS spectra of 4 to 27 amino acid peptides were composited with in silico digestion. MS/MS spectra of 2D LC-MS/MS were compared to the composite MS/MS spectra with Xcorr values above 2.0, 2.0, and 3.3 for 1+, 2+, and 3+ charge states of the peptide. Five MS/MS spectra with high intensity in each peptide of the candidate protein were selected from m/z 600–1250 for protein quantification.
Goto R., Nakamura Y., Takami T., Sanke T, & Tozuka Z. (2015). Quantitative LC-MS/MS Analysis of Proteins Involved in Metastasis of Breast Cancer. PLoS ONE, 10(7), e0130760.
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6

Quantitative LC-MS/MS Analysis of Peptides

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For LC-MS/MS analysis, dried peptides were reconstituted in 3 % acetonitrile/ 0.1 % formic acid (volume fraction) in water. Separation was performed on an Agilent Zorbax Eclipse Plus C18 RRHD column (2.1 mm × 50 mm, 1.8 μm particle) and MRM analysis was performed on an Agilent 6490 iFunnel Triple Quadrupole LC/MS system (Santa Clara, CA). Peptides were eluted at a flow rate of 200 μL/min using the following gradient of solvent B in solvent A: 3 % B for 3 min, 3 % to 30 % B in 30 min, 30 % to 50 % B in 5 min, and 50 % to 3 % B in 5 min. Solvent A was water containing 0.1 % formic acid (volume fraction) and solvent B was acetonitrile containing 0.1 % formic acid (volume fraction). The acquisition method used the following parameters in positive mode: fragmentor 380 V, collision energy 20 V, dwell time 100 ms, cell accelerator 4 V, electron multiplier 500 V, and capillary voltage 3500 V. MRM transitions for 2+ charge precursor ions and 1+ charge product ions were predicted using PinPoint software (Thermo Fisher Scientific, Waltham, MA).
Wang T, & Turko I.V. (2018). Proteomic toolbox to standardize the separation of extracellular vesicles and lipoprotein particles. Journal of proteome research, 17(9), 3104-3113.
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7

LC-MS/MS Peptide Quantification

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For LC-MS/MS analysis, dried peptides were reconstituted in 3% acetonitrile/0.1% formic acid (volume fraction). Separation was performed on an Agilent Zorbax Eclipse Plus C18 RRHD column (2.1 mm × 50 mm, 1.8 µm particle) and MRM analysis was performed on an Agilent 6490 iFunnel Triple Quadrupole LC/MS system (Santa Clara, CA). Peptides were eluted at a flow rate of 200 µL/min using the following gradient of solvent B in solvent A: 3% B for 3 min, 3% to 30% B in 30 min, 30% to 50% B in 5 min, and 50% to 3% B in 5 min. Solvent A was water containing 0.1% formic acid (volume fraction) and solvent B was acetonitrile containing 0.1% formic acid (volume fraction). The acquisition method used the following parameters in positive mode: fragmentor 380 V, collision energy 20 V, dwell time 100 ms, cell accelerator 4 V, electron multiplier 500 V, and capillary voltage 3500 V. MRM transitions for 2+ charge precursor ions and 1+ charge product ions were predicted using PinPoint software (Thermo Fisher Scientific, Waltham, MA).
Wang T., Anderson K.W, & Turko I.V. (2017). Assessment of Extracellular Vesicles Purity Using Proteomic Standards. Analytical chemistry, 89(20), 11070-11075.
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8

Quantitative Peptide Analysis by LC-MS/MS

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Dried peptides were reconstituted in 3% acetonitrile, 97% water, and 0.1% formic acid (volume fraction). Separation was performed on an Agilent Zorbax Eclipse Plus C18 RRHD column (2.1 mm × 50 mm, 1.8 μm particle) and multiple reaction monitoring (MRM) analysis was performed on an Agilent 6490 iFunnel Triple Quadrupole LC/MS system (Santa Clara, CA). Peptides were eluted at a flow rate of 200 μL/min using the following gradient of solvent B in solvent A: 3% B for 3 min, 3–30% B in 32 min, 30–50% B in 5 min, and 50–3% B in 3 min. Solvent A was water containing 0.1% (volume fraction) formic acid and solvent B was acetonitrile containing 0.1% formic acid. The acquisition method used the following parameters in positive mode: fragmentor 380 V, collision energy 20 V, dwell time 100 ms, cell accelerator 4 V, electron multiplier 500 V, and capillary voltage 3500 V. MRM transitions for 2+ charge precursor ions and 1+ charge product ions were predicted using PinPoint software (Thermo Fisher Scientific, Waltham, MA).
Anderson K.W., Mast N., Pikuleva I.A, & Turko I.V. (2015). Histone H3 Ser57 and Thr58 phosphorylation in the brain of 5XFAD mice. FEBS Open Bio, 5, 550-556.
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9

Hippocampal Prostaglandin Extraction and Quantification

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The extraction of prostaglandins was performed according to the method described in the previous paper39 (link). Briefly, brains were removed from the skull under deep anesthesia and the hippocampus was dissected on ice. The hippocampus was weighed and transferred to ice-cold methanol containing prostaglandin E2 and F2a standards (ratio 7:1:1, PGE2-d4 #314010 and PGF2a-d4 #316010: Cayman Chemical, Ann Arbor, MI, USA). Samples were homogenized by sonication and vortexing and centrifuged for 15 min at 10,000 g at 4 °C. Supernatants were then sampled, incubated for 30 min at − 80 °C and centrifuged at 1,000 g for 10 min. Supernatants were finally sampled and used for quantitative analysis by MS/MS liquid chromatography.
Samples were separated on EASY-nLC 1000 using NANOHPLC capillary column C18 (Nikkyo Technos, Tokyo, Japan) with a trap column and analyzed by triple-quadrupole MS (TSQ Vantage EMR: Thermo Fisher Scientific, Tokyo, Japan) in the Selected Reaction Monitoring (SRM) mode. Data were processed using PinPoint software (Thermo Fisher Scientific) for peak area calculation, using internal controls PGE2-d4 and PGF2a-d4 for normalization.
Arima-Yoshida F., Raveau M., Shimohata A., Amano K., Fukushima A., Watanave M., Kobayashi S., Hattori S., Usui M., Sago H., Mataga N., Miyakawa T., Yamakawa K, & Manabe T. (2020). Impairment of spatial memory accuracy improved by Cbr1 copy number resumption and GABAB receptor-dependent enhancement of synaptic inhibition in Down syndrome model mice. Scientific Reports, 10, 14187.
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10

Nano-LC-MS/MS Peptide Separation

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The pre-column was connected in-line with an analytical column (50µm ID × 10cm packed with 5µm beads (YMC gel, ODS-AQ, 12nm, S-5 µm, AQ12S05)) with integrated electrospray emitter tip [Martin 2000]. A 140 minute gradient from aqueous (0.2M acetic acid) and organic phase (0.2M HOAc, 70% MeCN) was used to separate peptides with a flow rate of 200 nL/min and a flow split > 99% and a spray voltage of 1800 V. Isolation widths of 0.7 were used for both Q1 and Q3 and collected for 0.05 seconds. A width of 0.01m/z was collected around each transition for quantification. Collision energies were determined per sequence with Thermo Pinpoint software.
Curran T.G., Zhang Y., Ma D.J., Sarkaria J.N, & White F.M. (2015). MARQUIS: A Multiplex Method for Absolute Quantification of Peptides and Post-Translational Modifications. Nature communications, 6, 5924.
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