TITLE: Laser Microdissection (LMD) for Regional Proteomics
AUTHORS: Samir Parikh, John Shapiro, Brad Rovin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Recent advances in multi-scale interrogation of human tissue, including advanced imaging techniques and the powerful application of large dataset “omics”, have displayed significant promise toward identifying new and specific therapeutic targets, predicting disease progression, and individualizing treatment in participants with acute and chronic kidney disease. Our site, formed by the alliance of Indiana University and Ohio State University, will implement an unbiased tissue interrogation workflow for KPMP human kidney biopsies that integrates largescale 3D tissue imaging for quantitative supervised and unsupervised analysis/cytometry with sub-segmental “omics” data on the same kidney biopsy specimen. The sub-segmental “omics” pipeline will use fluorescence based Laser MicroDissection (LMD) to isolate specific nephron segments and interstitial/other targeted areas, for downstream analysis with transcriptomics and proteomics. The omics analysis will be eventually expanded to include bulk epigenetics. Acting harmoniously with other KPMP sites, our interrogation techniques are expected to facilitate back-mapping of key molecular pathways to the biopsy, which can subsequently identify foci of injury and/or regeneration that can undergo targeted sampling to generate further enriched omics. Therefore, in addition to a significant contribution to the human kidney atlas, our approach will complement other interrogation techniques within KPMP by providing tissue context and increasing spatial resolution for molecular signatures that arise in heterogeneous areas during kidney disease.</div></div>

[STEPS]
?. [Cryosectioning for Regional Proteomics]
Sections are collected and mounted to LMD slides as specified in the  'Laser microdissection for regional transcriptomics and proteomics' protocol linked below. The LMD slides are then subsequently shipped from IU to OSU for analysis.
{"blocks":[{"key":"bg65u","text":"Recent advances in multi-scale interrogation of human tissue, including advanced imaging techniques and the powerful application of large dataset \u201comics\u201d, have displayed significant promise toward identifying new and specific therapeutic targets, predicting disease progression, and individualizing treatment in participants with acute and chronic kidney disease. Our site, formed by the alliance of Indiana University and Ohio State University, will implement an unbiased tissue interrogation workflow for KPMP human kidney biopsies that integrates largescale 3D tissue imaging for quantitative supervised and unsupervised analysis\/cytometry with sub-segmental \u201comics\u201d data on the same kidney biopsy specimen. The sub-segmental \u201comics\u201d pipeline will use fluorescence based Laser MicroDissection (LMD) to isolate specific nephron segments and interstitial\/other targeted areas, for downstream analysis with transcriptomics and proteomics. The omics analysis will be eventually expanded to include bulk epigenetics. Acting harmoniously with other KPMP sites, our interrogation techniques are expected to facilitate back-mapping of key molecular pathways to the biopsy, which can subsequently identify foci of injury and\/or regeneration that can undergo targeted sampling to generate further enriched omics. Therefore, in addition to a significant contribution to the human kidney atlas, our approach will complement other interrogation techniques within KPMP by providing tissue context and increasing spatial resolution for molecular signatures that arise in heterogeneous areas during kidney disease. ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Preparation of Frozen Sections for LMD]
70% Ethanol   X 2 (done at  )
4 °C
?. [Preparation of Frozen Sections for LMD]
Place slides in MS grad water for   X 2. Fresh MS grade water should be used with each wash.
?. [Preparation of Frozen Sections for LMD]
Stain slides with Hematoxylin QS (optional)
?. [Preparation of Frozen Sections for LMD]
Place slides in 70% ethanol for
?. [Preparation of Frozen Sections for LMD]
Place slides in 95% ethanol for
?. [Preparation of Frozen Sections for LMD]
Place slides in 100% ethanol #1 for
?. [Preparation of Frozen Sections for LMD]
Place slides in 100% ethanol #2 for
?. [Collection of LMD Tissue]
Make stock solution of NH3HCO3 1M or 0.5 M
Store in refrigerator for no more than 2 weeks and always look for precipitate. Presence of precipitate means it is time to make fresh stock.
?. [Collection of LMD Tissue]
For single 0.2 mg Rapigest tube to make final of 0.5%, make  of 50 mM NH3HCO3 add it to vial with Rapigest. Gently swirl  -  .
200 µl
Keep the solution refrigerated for no more than 2 weeks.
?. [Collection of LMD Tissue]
Use   of 0.5% Rapigest to collect each LMPC sample.
25 µl
?. [Collection of LMD Tissue]
Perform LMD on a Leica DFC7000T scope. Collect ~20,000 cells for each sample in under
?. [Collection of LMD Tissue]
After LMD collection, freeze in PCR tube using dry ice and then store at
-80 °C
?. [Trypsin Digestion]
Thaw samples in PCR tubes
?. [Trypsin Digestion]
Cut off the sides of the tube cap
?. [Trypsin Digestion]
Place   tubes into  tubes with the bottoms cut off. Apply pressure and secure with  pipette tip.
0.2 mL
1.5 mL
10 µl
?. [Trypsin Digestion]
Boil sample
?. [Trypsin Digestion]
Cool and add trypsin (trypsin is 200 ng/µl in 50 mM NH3HCO3 ) 1:30, briefly centrifuge sample  , then incubate overnight at
Centrifuge: 13000 33
37 °C
The amount of trypsin added is 1/30th the amount to total protein in the sample.
?. [Trypsin Digestion]
(Only if FFPE) Incubate sample at   for
60 °C
?. [Trypsin Digestion]
Add formic acid to final volume of 30% and make sure cloudiness is visualized
?. [Trypsin Digestion]
Incubate at  for
37 °C
?. [Trypsin Digestion]
Centrifuge for  at  and collect the supernatant without disturbing the pellet. Repeat two more times for a total of three centrifugations.
Centrifuge: 15000 33
?. [Trypsin Digestion]
Speedvac dry the sample and confirm presence of an amber pellet
?. [Trypsin Digestion]
Add  total of 2% acetonitrile and 0.1% formic acid mixture
20 µl
?. [Trypsin Digestion]
Vortex briefly, centrifuge briefly to bring all of the solution to the bottom of the tube.
?. [Trypsin Digestion]
Place the tube in an ice cold sonicating water bath for   then briefly centrifuge
?. [Trypsin Digestion]
Using Nanodrop spectrophotometer, check the peptide concentration using 280 absorbance. Use  on single orbitrap run with ~  gradient
1.5 µg
?. [Trypsin Digestion]
QA/QC: Determine if protein efficiency of recovery is > 50%
?. [HLPC and Mass Spectrometry]
Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis is performed  with an Easy-nLC 1000 coupled to an Orbitrap Eclipse mass spectrometer (Thermo Scientific, Waltham, MA).
?. [HLPC and Mass Spectrometry]
The LC system configured in a vented format consists of a fused-silica nanospray needle (PicoTip emitter, 75 μm inner diameter) (New Objective, Woburn, MA) packed in-house with 25 cm Magic C18 AQ 100 Å reverse-phase media (Michrom Bioresources, Auburn, CA) and a trap (IntegraFrit Capillary, 100 μm inner diameter) (New Objective, Woburn, MA) containing Magic C18 AQ 200 Å (2 cm).
?. [HLPC and Mass Spectrometry]
A measure of  of peptides are loaded onto the column from each sample and separated using a two-mobile-phase system consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The chromatographic separation is achieved over a 163-min gradient from 2 to 50% B (5–30% B for  , 30–50% B for  , and 50% B for  ) at a flow rate of 300 nl/min
1.5 µg
?. [HLPC and Mass Spectrometry]
The mass spectrometer is operated in a data dependent MS/MS mode over the m/z range of 400–1,500.  The mass resolution is set to 120,000. The automatic gain control target for the orbitrap is set to 2 × 105 with an injection time of 50 ms.
?. [HLPC and Mass Spectrometry]
For MS2, the quadrupole is used for isolation with a window of 1.6 m/z. The cycle time is set to 3 s, and the most abundant ions from the precursor scan with a charge state between 2 and 6 are selected for MS/MS analysis using 27% normalized HCD collision energy and analyzed with an ion trap. The automatic gain control target for the ion trap is set to 1 × 104 with an injection time of 90 ms. Selected ions are dynamically excluded for 20s.
?. [HLPC and Mass Spectrometry]
Data analysis is performed using Proteome Discoverer 2.2 (Thermo Scientific).
?. [HLPC and Mass Spectrometry]
Trypsin is set as the enzyme with maximum missed cleavages set to 2. The precursor ion tolerance is set to 10 ppm, and the fragment ion tolerance is set to 0.6 Da.
?. [HLPC and Mass Spectrometry]
Variable modifications are set to carbamidomethyl on cysteine residues, and oxidation of methionine residues.
?. [HLPC and Mass Spectrometry]
SEQUEST search results are run through Percolator for scoring. Identified peptides are filtered by a 1% peptide-level false discovery rate using q value of 0.01 from Percolator. In addition, false discovery rate at the protein level is calculated, and only the proteins with false discovery rate ≤1% are reported.
?. [Proteomics QC and OrbiTrap Eclipse]
The OrbiTrap Eclipse mass spectrometer is subjected to a mass calibration every week to ensure the instrument is optimized for obtaining high mass accuracy (
?. [Proteomics QC and OrbiTrap Eclipse]
The mass spectrometer is subjected to full calibration monthly to ensure that not only high mass accuracy is obtained, but to also ensure high sensitivity is obtained.
?. [Proteomics QC and OrbiTrap Eclipse]
HPLC function along with the mass spectrometer’s mass accuracy and resolution are tested by injecting a five-peptide standard (500 fmol of each peptide) into the LC-MS/MS system.
?. [Proteomics QC and OrbiTrap Eclipse]
HPLC retention times for each peptide should not deviate more than one minute from historic norms, mass accuracy should be less than 3 ppm, and mass resolution should be better than 120,000 at m/z 200.
?. [Proteomics QC and OrbiTrap Eclipse]
This five-peptide mixture is run on a daily basis and run intermittently during the analysis of large numbers of samples to verify the instrument’s performance at the beginning, middle, and end of large sample sets.
?. [Proteomics QC and OrbiTrap Eclipse]
The full LC-MS/MS data collection mode is tested after full instrument calibrations and before long sample sets by injection of   of a trypsin-digested yeast lysate (Promega).
200 ng
?. [Proteomics QC and OrbiTrap Eclipse]
A minimum of 6000 proteins should be detected for digest (less than 1% false discovery rate) after automated protein database searching.
?. [Proteomics QC and OrbiTrap Eclipse]
If peptide retention times, mass accuracy, or protein identification numbers do not meet defined criteria, the LC-MS/MS system is evaluated and steps are taken (e.g. cleaning of the mass spectrometer) to rectify the problem.
?. [Proteomics QC and OrbiTrap Eclipse]
Enrichment analysis to compare protein abundance to the set of maker panels specific to each nephron sub-segment.
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Collect paraffin sections cut 10 µm thickness in mass spec clean container with mass spec H2O (heat water in this container in the tissue water bath…do not heat water in microwave.
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
After air drying slides, place in desiccator with desiccant “stones” and under vacuum for 7 days
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Prepare all tubes with  each of required solutions
13 mL
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Place slides in 3 changes of octane  each occasionally inverting tube to help with deparaffinization
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Place slides in 3 changes of absolute ethanol  each with occasional gentle inversion
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Place slides in 1 change 90% absolute ethanol with occasional gentle inversion
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Place slides in 1 change 70% absolute ethanol  with occasional gentle inversion
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Place slides in H2O 2 changes  each with occasional gentle inversion
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Remove slides from H2O and stain with a few drops of hematoxylin for 2-5 seconds then immediately rinse in 2 changes of H2O 2 changes,  each with occasional gentle inversion
NOTE: All of the following steps are done with fresh solutions not used for the deparaffinization
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Place slides in 1 change 70% absolute ethanol  with occasional gentle inversion
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Place slides in 95% ethanol for  with occasional gentle inversion
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Place slides in 2 changes of absolute ethanol  each with occasional gentle inversion
?. [Cutting & Deparaffinization of FFPE Sections (FFPE Only, Protocol not active in KPMP)]
Remove slides and air dry for LMD
?. [Preparation of Frozen Sections for LMD]
(If staining with Hematoxylin) Place slide in MS grade water for  X 2