A STAGE Tip Procedure for Desalting and Purification of Peptides

Protocol

1. Purification and Desalting of Peptides

1. Pack three layers of C18 resin in a 200 μl pipette tip for each peptide sample to construct a STop And Go Extraction (STAGE)-tip.
2. Add 1/10 stopping solution (20% acetonitrile [ACN)] and 6% trifluoroacetic acid [TFA]) to samples.
3. Centrifuge samples for pellet precipitation at 13,200 rpm for 10 min.
4. Transfer the supernatant to a new 2 mL tube and discard the pellet.
5. Wash stage tips with 100 μl of 100% ACN.
6. Centrifuge STAGE tips at 1000 x g for 2 min.
   NOTE: Centrifuge STAGE tips until all liquid has run through the C18 resin.
7. Wash STAGE tips with 50 μl Buffer B (80% ACN, 0.5% Acetic acid, and ddH₂O).
8. Centrifuge STAGE tips at 1000 x g for 2 min.
9. Wash STAGE tips with 200 μl Buffer A (2% ACN, 0.1% TFA, 0.5% Acetic acid, and ddH₂O).
10. Centrifuge stage tips at 1000 x g for 2 min.
11. Load samples into STAGE tips.
12. Centrifuge samples at 1000 x g for 3-5 min.
13. Wash STAGE tips with 200 μl Buffer A.
14. Centrifuge at 1000 x g for 3-5 min.
15. Add 50 μl of Buffer B into each STAGE tip
16. Using a syringe, elute samples containing Buffer B into a 0.2 mL PCR strip tube.
17. Once samples are eluted, dry the peptides using a vacuum centrifuge for 45 mins.
18. Once samples are dry, store them at room temperature until MS.
    NOTE: If peptide storage is needed for more than two weeks, we suggest storing at -20°C.
19. Run the sample on a high-resolution mass spectrometry (MS).
    NOTE: Sample measurement parameters (e.g., amount of sample loaded, liquid chromatography gradient length, resolution) will depend on the instrumentation used.

2. Proteomic Profiling of Neutrophils 

1. Raw data processing
   1. Upload Raw data files obtained from MS into MaxQuant.
   2. The software is designed to run with bottom-up, data-dependent acquisition files, and many default parameters are optimized for such samples. Default parameters include Digestion (Trypsin P), Variable modifications of methionine oxidation (oxidation (M)) and N-acetylation of proteins (Acetyl (Protein N-term)), and fixed modifications of carbamidomethylation of cysteine (carbamidomethyl (C)).
   3. Adjustments to these parameters include label-free quantification (LFQ; set minimum ratio count to 1), Match Between Runs (i.e., on/off), and a minimum number of peptides identified per protein (recommend setting to 2).
   4. In global parameters, import the FASTA file for sequence identification. For this experiment, the Homo sapiens (Human) (UP000005640) FASTA file from the Uniprot database was used. NOTE: Record the following information for record-keeping: Organism ID = 9606; the number of proteins = 79,740; file download date.
   5. Set the number of computer cores to be used for processing and select Start.
   6. Upon completion of MaxQuant, a 'combined' folder, along with other files, will be generated.
      NOTE: The other files may be used to identify peptides, modifications, etc., and may be needed to upload the full dataset to data repositories, including Proteome Xchange (PRIDE).
2. Data analysis and visualization in Perseus
   1. Upload the 'proteingroups.txt' into Perseus and select all LFQ intensity files for the 'Main' window.
      NOTE: Baseline information is imported directly into the Perseus windows, but other information (e.g., modification sites) may need to be added manually.
   2. Filter rows by removing contaminants, reverse peptides, and peptides only identified by site.
   3. Transform the data by log₂(x).
   4. To observe the number of proteins detected in each replicate, construct a numeric Venn diagram.
   5. Set categorial annotations for each sample.
      NOTE: For statistical purposes, all replicates of one biological sample should have the same name.
   6. Filter rows based on valid values, with the protein identified in >50% of the replicates and within at least one group.
      NOTE: Filtering by valid values can be more stringent, depending on the experiment setup and goals (e.g., protein must be present in all samples).
   7. To replace missing values for LFQ, imputation from the normal distribution can be performed.
      ​NOTE: the default parameters are optimized for LFQ data but can be adjusted as desired.
   8. Add annotation information to the protein rows. Add the txt.gz FASTA file (obtained from the Perseus website) in the 'bin', 'conf', and 'annotation' folders and bring in specific terms (e.g., protein names, gene names, gene ontology).
   9. The matrix is now complete and can be used for statistical testing (e.g., ANOVA, t-tests) to determine significant changes in protein abundance between the tested conditions. Other forms of visualization tools include principal component analysis plots, heat maps, and category enrichment.
      NOTE: Annotation information can also be downloaded from UniProt and integrated into the Perseus framework.

Access restricted. Please log in or start a trial to view this content.

Materials

Name	Company	Catalog Number	Comments
15 ml Falcon Tube	VWR	470225-000
25 mL Serological	Fisher Scientific	14955235
Ammonium Bicarbonate (ABC)	ThermoFisher Scientific	A643-500
Acetic Acid	Sigma Aldrich	1099510001
Acetone	Sigma Aldrich	34850-1L
Acetonitrile (ACN), MS grade	Pierce	TS-51101
BSA assay kit	VWR	CA62408-940
C18 disks (STAGE tips)	Fisher Scientific	13110019
Centrifuge 5810 R	Sigma Aldrich	EP022628188
Dithiothreitol (DTT)	ThermoFisher Scientific	R0861	Prepare 1M stock of DTT aliquots and store at  -20°C
Fetal Bovine Serum (FBS)	ThermoFisher Scientific	12483020	Thaw out, prepare aliquots and stored at -20°C
HEPES	Sigma Aldrich	H3375	Prepare 8M Urea/40m HEPES aliquots, store at -20 °C
High-resolution mass spectrometer	ThermoFisher Scientific	726042
Iodoacetamide  (IAA)	Fisher Scientific	AAA1471514	Prepare 0.55M stock of IAA aliquots and store at  -20°C. Avoid light exposure
L-glutamine	Fisher Scientific	25030081	Thaw out, prepare aliquots and stored at -20°C
LoBind Microcentrifuge tubes	Eppendorf	13-698-794
MaxQuant	https://www.maxquant.org/
PCR strip tubes	VWR	76318-802
Penicillin Streptomycin, Amphotericin	Sigma Aldrich	A5955	Thaw out, prepare aliquots and stored at -20°C
Perseus	https://www.maxquant.org/Perseus/
phosphate-buffered solution (PBS)	Bioshop	PBS408	Store at room temperature
Probe sonicator	Fisher Scientific	100-132-894
Protease inhibitor cocktail (PIC) tablet	Sigma Aldrich	5892791001	Store at 4°C
RPMI 1640	ThermoFisher Scientific	11875093	Store at 4°C
Sodium Dodecyl Sulphate (SDS)	ThermoFisher Scientific	J18220.A1	prepare 2% SDS solution
SpeedVac Centrifuge	Eppendorf	07-748-15
STAGE tipping centrifuge	Sonation	STC-V2
T75 Flasks	ThermoFisher Scientific	156499
Trifluoroacetic acid	ThermoFisher Scientific	85183
Tris HCL pH 8.5	Fisher Scientific	BP152-1	Store at 4°C
Trypsin/LysC	Thermo Fisher Scientific	A40009	aliquot and store at -20°C
Ultrasonic bath	VWR	97043-960
Urea	Bioshop	URE001.1