Discovery Proteomics of Human Placental Tissue

Abstract

We describe a label-free proteomics protocol for the interrogation of the placental proteome. Step-by-step directions are provided from tissue clean-up and preparation, proteolytic digestion, nanoLC-MS/MS data collection and data analysis. The workflow has been applied towards exploring differential protein expression patterns in placentas from women who have been exposed to drugs during pregnancy relative to those who have not. We collected twenty tissue specimens, each representing a combination of spatially diverse sections across the placenta. These specimens were analyzed in the work described here, to survey information across the entire organ. This protocol can be scaled up or down as needed.

Introduction

A label-free proteomics workflow for placental tissue analysis is described, one which allows modelling and exploration of toxicant exposure during gestation. Placental tissue from women who have engaged in recreational drug use during pregnancy can be investigated to determine proteins affected by such exposure. Understanding the natural detoxification processes of the human placenta could potentially assist in identifying targets for prenatal therapies to reduce the effects of toxicants on developing foetuses. A label-free proteomic method has been developed to confidently identify a robust number of protein targets within each tissue sample. When considering under-studied systems such as the human placenta, and ailments associated with abnormalities in placental function, discovery-based proteomics can serve as an effective platform for future hypothesis-driven investigations of placental function. Nine different tissue sections from each placenta were combined to form one sample for the work described here, to survey information across the entire placenta. This protocol can be applied to a large amount of placental tissue (~1–2 grams). ^1–6 Although human tissue is specifically described in this protocol, this procedure may be used to study placental tissue from other mammals. However, the protein database against which the data is searched should be appropriate for the organism that is being studied.

• Label-free Discovery Proteomics

• Homogenization of >1 g placenta tissue

• Can be performed in ~3 days’ time, post placental tissue collection and sampling.

Materials

• Stainless steel beads (Fisher Scientific Catalog # 50-153-2314)

• Glass scintillation vials (Fisher Scientific Catalog # 03-341-25D)

• Vivacon-500, 30 kDa MWCO filters (Sartorius Catalog # VN01H22ETO)

• 1.5 mL Low Retention Eppendorf Tubes (Fisher Catalog # PI90410)

• 15 mL Falcon Tubes to prepare FASP solutions (Fisher Catalog # 05-527-90)

• 50 mL Falcon Tubes to prepare FASP solutions (Fisher Catalog # 14-959-49A)

• Oven, 37°C and 60°C (Fisher Catalog # 11-475-153)

• Acclaim PepMap™ C₁₈ trap column (Thermo Scientific Catalog # 164946)

• PepMap™ C₁₈ column (Thermo Scientific Catalog # ES802A)

• Autosampler vials and caps (Fisher Catalog # C4011-13 and 60180-676)

• Omni handheld homogenizer probe (Fisher Catalog # 15-340-167)

• Omni homogenizer tips (Fisher Catalog # 15-340-100)

• Fisherbrand Isotemp Shaker (Fisher Catalog # 02-217-741)

• IBI Scientific “The Belly Dancer” Orbital Platform Shaker (Fisher Catalog #15-453-211)

• Fisherbrand accuSkan FC Filter-Based Microplate Photometer (14-377-575)

• Thermo Scientific Sorvall Legend Micro 21 Microcentrifuge (Fisher Catalog #75-772-436)

• Eppendorf Microcentrifuge 5430R (Fisher Catalog #05-100-180)

• Fisherbrand Model 505 Sonic Dismembrator (Fisher Catalog FB5050110)

• Analytical balance

• Graduated cylinders, 10–500 mL are preferable

• Glass solvent bottle, 1 L

• Metal spatula to dispense solid reagents

• Weight boats to measure solid reagents

Chemicals

• Optima LC/MS Grade water (Fisher Scientific Catalog # W6-4)

• Optima LC/MS Grade ACN (Fisher Scientific Catalog # A955-4)

• Formic acid (Fluka Catalog # 94318-50ML)

• Ammonium bicarbonate (Millipore Sigma # 09830)

• Sodium deoxycholate (Millipore Sigma # D6750)

• Acetic acid (Fisher Scientific Catalog # 143-16)

• Hydrochloric acid (Fisher Scientific Catalog # 60-007-63)

• Dithiothreitol (BioRad Catalog # 161-0611)

• Iodoacetamide (BioRad Catalog # 163-2109)

• Urea (BioRad Catalog # 161-0730)

• Sequencing Grade Modified Trypsin (Promega Catalog # V5111)

• Pierce Standard LC/MS Grade BSA Protein Digest (Thermo Scientific Catalog # 88341)

• Pierce Standard HeLa Protein Digest (Thermo Scientific Catalog # 88329)

• BCA Protein Concentration Assay (Fisher Scientific Catalog # 23225)

• Roche Protease Inhibitor (Millipore Sigma Catalog # 04693116001)

• Halt Protease Inhibitor (Thermo Scientific Catalog # 78430)

• Phosphate buffer solution (Fisher Scientific Catalog # BP661-10)

• Pierce BCA Protein Assay Kit (Catalog # 23225)

Method

As with any proteomics sample preparation workflow, the samples, workspace, reagents and equipment should be safeguarded from keratin contamination. Keratin originates from skin and hair as well as dust, clothing and even latex gloves. If keratins are present in high concentrations, they can interfere with mass spectrometry analyses, rendering loss in signal and decreased identification of potentially interesting proteins. In the case of placental tissues, samples should also be adequately rinsed with ample quantities of sterile solution in order to minimize presence of maternal red blood cells (RBCs). Sterile water and phosphate buffered solution (PBS) with Halt protease inhibitor are used during various stages to ensure placental tissues are amply rinsed to minimize risk of contamination. Indeed, the issues with presence of haemoglobin from maternal RBCs can be similar to those with unwanted keratin presence in these proteomics samples. Workspaces and equipment should be kept clean, reagents should be prepared fresh (in clean containers) and gloves should be changed frequently. Leaning over the workspace must be avoided. Reading through the protocol in advance and pre-labelling any containers that are needed is recommended. Samples will change containers multiple times throughout the workflow and so keeping track of samples will be particularly important.

Collection of Tissues for Proteomic Analysis^7–9

A. Placental Collection and Storage

Once a study participant infant is delivered and the study team has confirmed with the maternal medical team that the placenta was not intended for pathology review, the study team collects the umbilical cord and placenta. Placentas should be placed in freezer-safe plastic containers or wrapped in aluminum foil and placed into a plastic freezer bag for temporary storage at 4°C. Samples are transferred to −80°C within 24 hours, in keeping with data from Sjaarda et al., 2018 who determined that refrigeration for up to 24 hours prior to processing/storage does not compromise results. Placentas are kept in secure storage at −80°C until time of sampling. Given the often unpredictable timing of placenta availability and the need to ensure timely collection and storage, it is important to designate key personnel and procedures well in advance in order to maximize efficiency and safeguard the integrity of samples. For the results reported in this study, a total of 64 placental samples were collected between September 2017 and August 2019. Of these, 20 samples (including 7 exposed and 13 controls) met inclusion criteria and were subsequently analyzed using the proteomics workflow detailed here.

Processing Time: 30– 60 minutes per placenta

Frozen samples are removed from −80°C and allowed to thaw at 4°C for approximately 18–22 hours. Placentas are placed with umbilical cord side up on a stainless-steel tray placed on top of frozen steel beads to keep tissues cold during processing. Punch biopsies are obtained in a 3×3 grid pattern from the left, middle, and right sides of the placenta per 3 rows, for a total of 9 biopsy samples to represent centrally and peripherally located tissues based on the systematic uniform random approach suggested by Mayhew, 2008. This sufficiently exceeds the minimum 4 samples per placenta to generate representative data suggested by Burton et al., 2014. Each sample is taken with a 6 mm diameter biopsy punch. However, since placentas are different thicknesses, each core varies in mass between approximately 1.2 to 3.8 g. Core samples are rinsed three separate times in PBS with Halt protease inhibitor on ice for 5 minutes each to remove maternal blood and leukocytes, possible confounders for proteomic analysis.⁹ All nine core samples from each placenta are placed into glass scintillation vials. Samples are stored at −80°C until they are processed.

Preparation of Placental Tissues for Proteomic nanoLC-MS/MS Analysis

Time Commitment: (2–3 days)

A. Preparation of Buffers Prior to Tissue Processing [A]

Processing time: 30–45 minutes

Buffers should be made fresh on the day of the sample preparation step. To prepare in advance, only measure out dry reagents and make appropriate solutions on the morning of sample preparation.

B. Preparation of Reducing and Alkylating Agent Aliquots prior to Proteolytic Digestion [A]

Processing time: 30–45 minutes

C. Preparation of Protein Solubilisation Buffers prior to Proteolytic Digestion [A]

Processing time: 45–60 minutes

Buffers should be made fresh on the day of digestion, to maintain buffer strength. To prepare in advance, only measure out dry reagents and add water on the morning of digestion.

D. Safety Precautions

BSL2 facilities were used and all contaminated materials and waste were appropriately autoclaved before disposal. Standard laboratory Personal Protective Equipment (PPE) should be worn during these experiments; this includes safety glasses, a clean lab coat or disposable gown, and disposable gloves. Since human tissues were involved, we also recommend masks and hair covers as part of standard PPE. This also minimizes risk of keratin contamination of samples. Homogenization/sonication of tissues should take place under a hood. Upon completion of sampling, excess tissue samples should be appropriately disposed of via approved human tissue waste disposal routes only.

E. Tissue Preparation (1–2 days) [A]

Processing time: 9–10 hours

* End of Day 1 out of 3 *

For a 3-day preparation, consider completing this step separately. This is recommended for larger quantities of samples (> 10). It can be done right after tissues are collected/received. For a 2-day preparation, consider completing as a first step and following with homogenization. All buffers and tissues should be kept on ice in order to prevent degradation of the tissue and indiscriminate cleavage of proteins.

* End of Day 2 out of 3 (or Day 1 out of 2 if tissue washing/homogenization steps are combined) *

F. Filter-Aided Sample Preparation (FASP) and Proteolytic Digestion^5,6 [A]

Processing time: 9–10 hours

The remaining procedure should be completed within the same day. The procedure takes a minimum of 7 hours. If using one oven for the entire workflow, make sure that it is set to 60°C prior to beginning. Gloves should always be used and replaced often throughout procedure to avoid contamination. Long hair should be pulled back to minimize keratin contamination. Leaning over the samples and workspace should be avoided to further help with minimizing keratin impurities.

**Change Collection Tubes Under Filter**

All eluents should be kept from this point forward in collection tubes labelled “peptides.” Waste collection tubes can be discarded.

* End of Day 3 out of 3 (or Day 2 out of 2) *

Samples are now ready for nanoLC-MS/MS analysis. An aliquot (~20 μL) of each sample can be added to an appropriately labelled LC-MS autosampler vial and placed in the nanoLC autosampler for analysis. The remaining sample can be stored at −20°C. Two microliter injections are performed during nanoLC-MS/MS experiments.

G. Separation and Analysis by nanoLC-MS/MS [A]

Sample order should be randomized prior to sequencing to avoid bias from sequencing order. See section on “Quality Control” for recommendations on interspersed quality control and blank sample sequencing. Each sample should be subjected to online desalting and reversed-phase nano-LC separation (in a “trap and elute” configuration.) Separated peptides are ionized via electrospray for mass spectrometry analysis. A data dependent acquisition method for untargeted or discovery analysis should be used. The attached files provide recommended settings for a two-hour analytical separation and DDA analysis, with a 140-minute total method length, using an EASY nano-LC system coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).

Tip: It is helpful to find a systematic way of loading the samples into the autosampler during longer experiments. This prevents degradation of samples that are analyzed later from sitting in the autosampler for more than a few days.

Quality Control (QC)

• Randomized samples, BSA QC (std. BSA digest) and blanks (MPA) are run as follows: blank, BSA QC, blank, Sample-01, Sample-02, Sample-03, Sample 04, blank, BSA QC, blank, Sample-05, Sample-06, Sample-07, Sample-08, blank, BSA QC, blank… etc. Through the AutoQC software on PanoramaWeb (https://panoramaweb.org/home/), BSA QCs enable the monitoring of such metrics as retention time reproducibility, peak area and FWHM in real time.⁷ A guide set should first be created (prior to any experiments) from five- seven independent BSA runs with each trap column, analytical column, or solvent change to account for shifts in retention times from these factors. Metrics are expected stay within three standard deviations from the guide set mean value. If these metrics fall outside of three standard deviations or are not reproducible, this could indicate an issue with instrument performance. For example, if major shifts in peptide retention times are visible, this may indicate an issue with the nLC system or columns. Users should troubleshoot the system as needed and consider re-analyzing biological replicates if metrics indicate system deterioration during the experiment. The importance of monitoring system suitability in bottom-up proteomics experiments is discussed further by Bereman and coworkers.¹⁰

• Several standard HeLa digest runs (HeLa QC) are conducted at the beginning and end of experiments to check on proteome coverage. Significant drops in coverage between runs may indicate a deterioration in system suitability.

Label-Free Data Analysis

Data files are now ready to be analyzed using software such as Proteome Discoverer for identification and quantification of proteins. The attached analysis workflow files for use with Proteome Discoverer 2.4 software are summarized in the following figures and tables. The basic parameters listed can be used with alternative search software. The identification of haemoglobin protein may be checked to evaluate degree of interference. While the data shared here is in raw abundance format (i.e., unnormalized) quantification methods should be carefully considered before drawing statistical and biological conclusions from such a dataset. Normalization methods are available in Proteome Discoverer 2.4 (e.g., to the total peptide amount in the Precursor Ions Quantifier node.) Open-source tools such as NormalyzerDe are available to assist in normalization method selection and dataset analysis¹¹.

Figure 1.

Processing workflow tree

Figure 2.

Consensus workflow tree.

Table 1.

Rinsing Solution, Protease Inhibitor in Water

Processing time	Step	Comments / Tips
15–30 minutes	Measure out water scaled to appropriate amount needed. At least 25 mL per sample will be needed, but if tissues appear very bloody, more might be needed. High quantities of haemoglobin from blood can dominate mass spectrometry signal and diminish identifications of other interesting proteins. Add to glass solvent bottle.	Scaling Example: It is a good idea to prepare additional volume for ample supply. For 5 washes of 20 samples: 5 mL × 5 rounds × 20 samples + 50 mL extra volume = 550 mL
	Add one Roche Protease Inhibitor Tablet for every 50 mL water per manufacturer’s instructions. Shake until dissolved.	If samples are very bloody, additional rinse steps can be applied. For example, it might take 10 wash steps (50 minutes total shaking, 50 mL rinsing solution per sample) to rinse out most of the blood from the sample.
	Rinsing solution should always be kept on ice.

Table 2.

Lysis Buffer, 50 mM ABC and 1% SDC (w/v) with Protease Inhibitor, 500 mL

Processing time	Step	Comments / Tips
15–20 minutes	Measure out 2.0 ± 0.1 g ABC and add to glass solvent bottle.	If necessary, this step can be scaled down to preserve reagents. The following should be considered when scaling:  □ 10 mL per sample to submerge sample tissue (more or less, depending on amount of sample)  □ Volume needed as a blank for protein quantitation method  □ Volume need to rinse sonicator probe (∼ 3 mL per sample to prepare 3 fresh tubes for rinsing)
	Measure out 5.0 ± 0.1 g SDC and add to same glass solvent bottle.
	Add 500 mL of water to glass solvent bottle. Shake well until reagents are fully dissolved.
	Add 10 (one for every 50 mL buffer) Roche Protease Inhibitor Tablets. Shake until dissolved.
		Note that lysis buffer should always be kept on ice.

Table 3.

Reducing Agent, Dithiothreitol (500 mM)

Processing time	Step	Comments / Tips
15–30 minutes	Weigh out 77.1 ± 0.1 mg of dithiothreitol into an Eppendorf tube.	Prepared DTT solution can be stored at −20°C for at least 1 month.
	Reconstitute in 1 mL of water.
	Vortex.
	Aliquot solution into Eppendorf tubes in 20 μL volumes and store at −20°C until ready for use.

Table 4.

Alkylating Agent, Iodoacetamide (500 mM)

Processing time	Step	Comments / Tips
15–30 minutes	Prepare in as little light as possible. Compound is sensitive to light.	Prepared IAA solution can be stored at −20°C for at least 1 month.
	Weigh out 305 ± 0.1 mg of iodoacetamide into a 15 mL Falcon tube.
	Reconstitute in 3.3 mL of water.
	Vortex.
	Aliquot into Eppendorf tubes (volumes appropriate for a 6 μL aliquot per sample, between 30 μL and 150 μL for example) and store at −20°C in the dark until ready for use.

Table 5.

50 mM ABC with 1% (w/v) SDC

Processing time	Step	Comments / Tips
15–30 minutes	Measure out 79.1 ± 0.1 mg ABC and add this to a 50 mL Falcon Tube.	Carefully tip the Falcon Tube on its side before adding water. This prevents SDC from getting stuck in the bottom of the Falcon Tube and helps it to dissolve completely. If necessary, centrifugation can help minimize bubbles from SDC.
	Measure out 200.0 ± 0.1 mg SDC and add to same Falcon Tube containing ABC.
	Add approximately 10 mL water and vortex until dissolved. Allow bubbles to settle.
	Add water until solution reaches the 20 mL mark on the Falcon Tube. Vortex and allow bubbles to settle.

Table 6.

50 mM ABC only

Processing time	Step	Comments / Tips
15–30 minutes	Measure out 79.1 ± 0.1 mg ABC into a 50 mL Falcon Tube.	The pH of ABC buffers in this protocol should be about 7.8.
	Add approximately 5 mL water and vortex until fully dissolved.
	Add water until solution reaches the 20 mL mark on the Falcon Tube.

Table 7.

50 mM ABC and 8M Urea

Processing time	Step	Comments / Tips
15–20 minutes	Measure out 79.1 ± 0.1 mg ABC into 50 mL Falcon Tube.	This will seem like a large volume of urea and will take a lot of time to fully dissolve. Add water carefully and slowly to avoid adding over the 20 mL point. Warming in your hands will help with dissolution. Do not heat the solution.
	Measure out 9.8 ± 0.1 g urea into same Falcon Tube.
	Add 10 mL water and vortex until mostly dissolved.
	Bring up to 15 mL mark with water. Vortex for a few minutes until mostly dissolved.
	Add water until solution reaches the 20 mL mark on the Falcon Tube.

Table 8.

Rinsing Blood from Samples

Processing time	Step	Comments / Tips
2–3 hours	Prepare rinsing solution fresh on day of use (see above, Table 1). Solution should always be scaled based on the number of samples and kept on ice after preparation.	This can be done on a separate day. Depending on how many samples are being processed or how many rinses are required to remove as much visible blood as possible, this step may require longer net processing time.
	Add 5 mL protease inhibitor solution to each sample. Samples should be kept on ice throughout entire washing procedure.
	Shake samples on BellyDancer shaker at the highest speed possible while on ice for 5 minutes.
	Replace inhibitor solution with fresh 5 mL, and repeat process five times for a total of 25 minutes of shaking (or more as needed, see tip).

Table 9.

Tissue Homogenization

Processing time	Step	Comments / Tips
3–4 hours	Prepare lysis buffer (see above, Table 2). Lysis buffer should be kept on ice at all times.	This step should take place under a hood. This can be done on a separate day. Depending on how many samples are being processed, this step may require longer net processing time.
	Add 10 mL of cold lysis buffer to each tissue container. Tissue should be submerged in buffer. Tissues should be kept on ice.
	Using the Omni handheld homogenizer under a hood, homogenize each sample individually on ice for 4–5 minutes until sample appears homogeneous in solution. Highest speed (35,000 rpm) is recommended for large amounts of tissue.
	Replace with a fresh homogenizer probe before the next sample is processed. Place the samples on ice after homogenization.

Table 10.

Tissue Sonication

Processing time	Step	Comments / Tips
2–3 hours	While on ice, probe sonicate each tissue at 20% power for 2 minutes using the Fisherbrand Model 505 Sonic Dismembrator. Sample should appear more homogenous.	This step should take place under a hood. Homogenized samples tend to settle and separate while sitting on ice. Sonication is important to ensure a homogeneous aliquot of tissue homogenate is removed from the sample for analysis.
	Aliquot approximately 1.0–1.5 mL sonicated sample into an Eppendorf tube. Immediately place on ice.
	In between samples, clean probe with methanol, followed by three clean tubes of fresh lysis buffer.

Table 11.

Centrifugation and Removal of Lysate

Processing time	Step	Comments / Tips
2–3 hours	Centrifuge all samples at 16,000 × g for 30 minutes at 4°C to form pellets.	With high quantities of tissue in 10 mL lysis buffer, concentration risks being higher than the calibration curve of the Pierce BCA Protein Assay Kit. It is therefore recommended to create a 1:10 diluted sample in Lysis Buffer prior to the total protein quantification assay analysis.
	Remove lysate to a fresh tube and label. Care should be taken not to disturb the pellet. Lysates should be kept on ice, and pellets can be stored at −80°C for potential future analysis, if needed. Lysates of samples are now ready for protein quantitation and digestion.
	Quantitation of protein can be performed using the Pierce BCA Protein Assay Kit or using the Nanodrop A280 protein assay. Lysis buffer should be used as a blank. Following protein quantitation, lysates should be stored at −80°C until the beginning of the digestion procedure.

Table 12.

Aliquoting and Diluting

Processing time	Step	Comments / Tips
30–45 minutes	Remove samples from freezer. Once thawed, aliquot volume containing 50 μg of sample into separate Eppendorf Tubes based off measured protein concentrations. This is important to normalize sample protein for the purpose of performing relative quantification of specific proteins detected in nanoLC-MS/MS analysis.	With large numbers of samples, it is very important to keep track of labelling. Samples will change containers a few times in this protocol.
	Bring samples up to 200 μL final volume with buffer containing 50 mM ABC and 1% (w/v) SDC to solubilize protein. Vortex.

Table 13.

Denaturing Proteins by Breaking Disulfide Bonds between Cysteine Residues

Processing time	Step	Comments / Tips
45–60 minutes	Add 180 μL buffer containing 500 mM ABC and 1% (w/v) SDC to thawed, 20 μL aliquot of DTT. If using more than one aliquot, combine into one Eppendorf tube and vortex. The DTT solution has now been diluted to 50 mM.	Prepare one DTT aliquot per 9 samples to account for any volume lost in pipetting steps.
	Add 20 μL of 50 mM DTT to each sample to yield a final concentration of 4.5 mM DTT. Vortex each sample.
	Incubate samples at 60°C for 30 minutes.
	Remove from oven and allow to cool to room temperature before continuing.

Table 14.

Alkylating Reactive Thiols to Prevent Reformation of Disulfide Bonds

Processing time	Step	Comments / Tips
30–45 minutes	If using more than one 500 mM IAA aliquot, combine into one Eppendorf tube and vortex.	Perform alkylation in as low of light as possible. IAA is light sensitive. While samples are incubating with DTT, remove IAA aliquots from freezer and allow to thaw in the dark. Thaw enough aliquots for 6 μL per sample while accounting for any sample lost during pipetting steps (for example, thaw 6 μL × # samples + 5–10 μL).
	Add 6 μL IAA to each sample to yield a final IAA concentration of 15 mM. Vortex.
	Incubate samples at room temperature in the dark for 20 minutes.

Table 15.

Transferring Samples to MWCO Filtration Units

Processing time	Step	Comments / Tips
15–30 minutes	Label a Vivacon-500 30 kDa MWCO filtration unit (filter and tube) for each sample and passivate by adding 20 μL 50 mM ABC + 1% SDC to the top of each filter.	Prepare (label and passivate) filtration units while samples are undergoing 20-minute alkylation reaction. The first collection tubes should be labelled “waste” as these will contain small molecules being washed from protein samples. A second set of collection tubes should be labelled “peptides” and set aside for post-digestion collection. Note that passivating MWCO filters before use is important to prevent protein loss in the FASP workflow.
	Transfer entire volume of each sample to appropriate filtration unit.
	Centrifuge sample-containing filtration units at 12,000 × g for 15 minutes.

Table 16.

Washing Twice with Buffer Containing 50 mM ABC and 8 M Urea

Processing time	Step	Comments / Tips
1–2 hours	Add 200 μL 50 mM ABC + 8 M urea to each sample filtration unit. Centrifuge units for 15 minutes at 12,000 × g.	There are many steps requiring centrifugation. It is helpful to check off which steps have already been completed to keep track. Keeping an eye on the level of eluted solution can also be helpful since waste should be emptied every two spin cycles (so waste should be emptied a total of 2 times before switching to final collection tubes.). Sometimes, due to filter clogging, the filtration rate may be rather slow. In such a situation, it may be ok to increase the centrifugation speed (for any of the steps requiring centrifugation) to 12,500 × g or 13,000 × g, but no more. A number of washing steps are encountered in this workflow. Based on the buffer being used, these steps help to clean and denature the protein, getting the sample ready for proteolytic digestion. Note that some buffer components, like urea, are great for protein denaturation but must be removed from the samples prior to proteolytic digestion to prevent inhibition of the protease.
	Empty eluent into waste so that it does not reach a volume that touches the filter. Handle the filter very carefully.
	Repeat the wash with another 200 μL 50 mM ABC + 8 M urea buffer. Centrifuge units for 15 minutes at 12,000 × g.

Table 17.

Washing Twice with Buffer Containing 50 mM ABC

Processing time	Step	Comments / Tips
1–2 hours	Add 200 μL 50 mM ABC (only ABC) to each sample filtration unit. Centrifuge units for 15 minutes at 12,000 × g.	Reconstitute trypsin during last wash step. Particularly for the last wash step, increase the centrifugation speed to 12,500 × g or 13,000 × g if need be to pass all the wash solution through the filter.
	Empty eluent into waste for the second time so that it does not reach a volume that touches the filter. Handle the filter very carefully.
	Repeat the wash with another 200 μL 50 mM ABC (only) buffer. Centrifuge units for 15 minutes at 12,000 × g.

Table 18.

Proteolytic Digestion

Processing time	Step	Comments / Tips
15–30 minutes + 4 hours shaking time	Reconstitute trypsin in 200 μL 0.01% acetic acid to stabilize protease and yield a 0.1 μg/μL protease concentration. Vortex.	Wrap a small amount of parafilm around tops of filtration units to prevent them from popping open during the 4-hour digestion period. Remove the parafilm before centrifugation (following digestion).
	Add 70 μL 50 mM ABC buffer to each filtration unit.
	Add 10 μL trypsin solution directly to ABC volume on top of filter for a 1:50 enzyme to protein ratio. Do not pipette on to the sides of the filter.
	Shake filtration units at 600 rpm at 37°C for 4 hours on Isotemp Shaker.

Table 19.

Elution of Tryptic Peptides

Processing time	Step	Comments / Tips
45–60 minutes	Centrifuge filtration units for 15 minutes at 12,000 × g to elute peptides.	To maximize tryptic peptide recovery, increase the centrifugation speed to 12,500 × g or 13,000 × g if need be to pass all the digest solution through the filter.
	Add 15.8 μL 50 mM ABC to elute any remaining peptides on filter. Centrifuge for 15 min at 12,000 × g.

Table 20.

Quenching of Enzyme

Processing time	Step	Comments / Tips
15–30 minutes	Discard filters.	Acidifying the peptide solution quenches the trypsin protease.
	Add 4.2 μL of 6 M HCl to each sample.

Table 21.

nanoLC Parameters

Sample pickup and Loading
Sample Pickup
Volume	2	μL
Flow	10	μL/min
Sample Loading
Volume	6	μL
Flow	1	μL/min
Max. Pressure	700	Bar
Solvents	A: water/acetonitrile + formic acid (98/2 + 0.1 % by volume)	B: water/acetonitrile + formic acid (80/20 + 0.1 % by volume)
Gradient
Time	Duration	Flow (nL/min)	%B
0:00	0:00	300	5
2:00	2:00	300	5
107:00	105:00	300	25
122:00	15:00	300	40
123:00	1:00	300	95
124:00	1:00	300	95
140:00	16:00	300	95
Pre-column and Analytical Column
Pre-column Equilibration
Volume	3	μL
Flow	3.00	μL/min
Max. Pressure	700	Bar
Analytical Column Equilibration
Volume	6	μL
Flow	-	μL/min
Max. Pressure	700	Bar

^*Custom wash used for Autosampler and column heating controlled externally on Easy-Spray Ion Source. Column is kept at 45 °C throughout analysis.

Table 22.

Mass Spectrometric Parameters (Data Dependent Acquisition)

Ion Source Properties
Ion Source Type	NSI
Spray Voltage	Static
Positive Ion (V)	1800
Gas Mode	Static
Sweep Gas (Arb)	0
Ion Transfer Tube Temp (°C)	275
FAIMS Mode	Not Installed
Tune Settings
Application Mode	Peptide
Method Duration	140
Infusion Mode	Liquid Chromatography
Expected LC Peak Width (s)	15
Advanced Peak Determination	Checked
Default Charge State	2
Internal Mass Calibration	User-defined Lock Mass
Current Lock Mass for Positive Ion Mode	445.12006
Method
Time Range (min)	0–140
Full Scan	2 sec
MIPS
Intensity
Charge State
Dynamic Exclusion
ddMS2
Full Scan Properties
Orbitrap Resolution	120000
Scan Range (m/z)	375–1600
RF Lens (%)	40
AGC Target	Custom
Normalized AGC Target (%)	300
Maximum Injection Time Mode	Custom
Maximum Injection Time (ms)	100
Microscans	1
Data Type	Profile
Polarity	Positive
Source Fragmentation	Unchecked
MIPS Properties
Monoisotopic peak determination	Peptide
Relax restrictions when too few precursors are found	Checked
Intensity Properties
Filter Type	Intensity Threshold
Intensity Threshold	5.00E+03
Charge State Properties
Include charge state(s)	2–6
Include undetermined charge states	unchecked
Dynamic Exclusion Properties
Dynamic Exclusion Mode	Custom
Exclude after n times	1
Exclusion duration (s)	20
Mass Tolerance	ppm
Low	10
High	10
Exclude isotopes	checked
Perform dependent scan on single charge state per precursor only	checked
Data Dependent MS2 Scan Properties
Multiplex ions	unchecked
Isolation Window (m/z)	1.4
Isolation Offset	Off
Collision Energy Mode	Fixed
Collision Energy Type	Normalized
HCD Collision Energy (%)	30
Orbitrap Resolution	7500
TurboTMT	Off
Scan Range Mode	Define First Mass
First Mass (m/z)	100
AGC Target	Custom
Normalized AGC Target (%)	100
Maximum Injection Time Mode	Custom
Maximum Injection Time (ms)	18
Microscans	1
Data Type	Centroid

Table 24.

Processing Workflow Nodes and Basic Parameters

Spectrum Files RC
Search Settings
Protein Database	Contaminants.fasta; Homo Sapiens (Swiss Prot TaxID=9606)
Enzyme Name	Trypsin (Full)
1. Static Modification	Carbamidomethyl/ +57.021 Da (C)
Spectrum Selector
General Settings
Precursor Selection	Use MS1 Precursor
Provide Profile Spectra	TRUE
Spectrum Properties Filter
Min. Precursor Mass	350 Da
Max. Precursor Mass	5000 Da
Peak Filters
S/N Threshold (FT-only)	1.5
Sequest HT
Input Data
Protein Database	Contaminants.fasta; Homo Sapiens (Swiss Prot TaxID=9606)
Enzyme Name	Trypsin (Full)
Max. Missed Cleavage Sites	2
Min. Peptide Length	6
Max. Peptide Length	144
Tolerances
Precursor Mass Tolerance	5 ppm
Fragment Mass Tolerance	0.02 Da
Spectrum Matching
Weight of a Ions	0.2
Weight of b Ions	1
Weight of x ions	0.2
Weight of y ions	1
Dynamic Modifications
Max. Equal Modifications Per Peptide	2
Max. Dynamic Modifications Per Peptide	3
1. Dynamic Modification	Oxidation/ +15.995 Da (M)
2. Dynamic Modification	Deamidated/ +0.984 Da (N, Q)
Dynamic Modifications (protein terminus)
1. N-Terminal Modification	Acetyl/ +42.011 Da (N-terminus)
2. N-Terminal Modification	Met-loss + Acetyl/ −89.030 Da (M)
Static Modifications
1. Static Modification	Carbamidomethyl/ +57.021 Da (C)
Percolator
Target/Decoy Strategy
Target/Decoy Selection	Concatenated
Validation based on	q-Value
Input Data
Maximum Delta CN	0.05
FDR Targets
Target FDR (Strict)	0.01
Target FDR (Relaxed)	0.05

Table 25.

Consensus Workflow Nodes and Basic Parameters

MSF Files
Storage Settings
Spectra to Store	Identified or Quantified
Feature Traces to Store	All
PSM Grouper
Peptide Group Modifications
Site Probability Threshold	75
Peptide Validator
General Validation Settings
Validation Mode	Automatic (Control peptide level error rate if possible)
Target FDR (Strict) for PSMs	0.01
Target FDR (Strict) for Peptides	0.01
Peptide and Protein Filter
Peptide Filters
Peptide Confidence At Least	High
Keep Lower Confident PSMs	FALSE
Protein Filters
Minimum Number of Peptide Sequences	1
Protein Grouping
Protein Grouping
Apply strict parsimony principle	TRUE
Peptide in Protein Annotation
Flanking Residues
Annotate Flanking Residues of the Peptide	TRUE
Modifications in Peptide
Protein Modifications Reported	Only for Master Proteins
Modifications in Protein
Modification Sites Reported	All And Specific
Minimum PSM Confidence	High
Report Only PTMs	True
Positions in Protein
Protein Positions for Peptides	Only for Master Proteins
Feature Mapper
Chromatographic Alignment
Perform RT Alignment	TRUE
Maximum RT Shift [min]	2
Feature Linking and Mapping
Minimum S/N Threshold	3
Precursor Ions Quantifier
General Quantification Settings
Peptides to Use	All
Precursor Quantification
Precursor Abundance Based On	Area
Normalization and Scaling
Normalization Mode	Total Peptide Amount
Exclude Peptides from Protein Quantification
For Protein Roll-Up	Use All Peptides
Quan Rollup and Hypothesis Testing
Protein Abundance Calculation	Summed Abundances
N for Top N	5
Protein Annotation
Annotation Aspects
1. Aspect	Biological Process
2. Aspect	Cellular Component
3. Aspect	Molecular Function
Protein Marker
Contaminant Database
Protein Database	Contaminants.fasta
Protein FDR Validator
Confidence Thresholds
Target FDR (Strict)	0.01
Target FDR (Relaxed)	0.05

Table 26.

Summary of Protein Identification Results

Sample	Proteins	High Confidence Proteins	Quantified Proteins	Peptides	PSMs	MS/MS
1	3,144	2,775	2627	23,375	40,593	222,373
2	3,648	3,178	3021	27,178	44,909	225,954
3	3,310	2,937	2788	24,300	42,360	221,100
4	3,267	2,853	2718	23,891	41,239	220,129
5	3,361	2,872	2757	25,485	43,301	217,922
6	3,726	3,266	2088	27,736	45,731	225,942
7	3,376	2,957	2803	24,598	41,500	222,897
8	3,034	2,651	2520	22,169	39,336	224,590
9	3,649	3,166	2982	27,433	45,999	224,925
10	3,225	2,779	2642	24,077	43,041	219,667
11	3,734	3,333	3150	27,411	44,724	224,688
12	3,544	3,110	2938	25,842	43,169	223,632
13	3,706	3,251	3068	27,647	45,761	225,482
14	3,763	3,302	3110	27,789	45,395	224,921
15	3,372	2,938	2779	24,681	43,730	223,157
16	3,285	2,883	2744	24,323	42,589	226,070
17	3,512	2,992	2842	25,502	43,077	224,773
18	3,788	3,324	3145	27,998	46,399	226,754
19	3,158	2,798	2622	22,823	37,952	219,531
20	3,402	2,966	2837	24,998	43,694	223,508
Average	3,450	3,017	2809	25,463	43,225	223,401
Standard Deviation	233	209	251	1,826	2,280	2,530
%CV	6.8	6.9	8.9	7.2	5.3	1.1

Table 27.

Summary of Haemoglobin Identification across Samples

	Peptides	PSMs
Haemoglobin Sum (averaged over 20 replicates)	107 +/− 8	1,996 +/− 366
Total Sum (averaged over 20 replicates)	25,463 +/− 1826	43,225 +/− 2,280
% of Total Identifications	0.42%	4.60%

Acronyms and Abbreviations

ABC

Ammonium bicarbonate

AGC

Automatic gain control

BCA

Bicinchoninic acid

DDA

Data dependent acquisition

DTT

Dithiothreitol

FASP

Filter Aided Sample Preparation

HCD

Higher-energy collision-induced dissociation

HCl

Hydrochloric acid

HCOOH

Formic acid

IAA

Iodoacetamide

MPA

Mobile phase A

MPB

Mobile phase B

MWCO filter unit

Molecular weight cut off filter unit

PBS

Phosphate buffered solution

PD

Proteome Discoverer

QC

Quality Control

RBC

Red blood cells

SDC

Sodium deoxycholate

[A]

signifies an important step in this protocol where alternative methods exist that may be used but were not tested here