Acetonitrile (ACN) and methanol were purchased from Burdick and Jackson (Muskegon, MI, USA), and trifluoroacetic acid (TFA) and ammonium bicarbonate (NH4HCO3) were purchased from J. T. Baker (Phillipsburg, NJ, USA). The standard peptide Cys-Kemptide (CLRRASLG, [M+H]+
m/z 875.49) was purchased from American Peptide Co. (Sunnyvale, CA, USA). Iodoacetamide (IAA), tetrahydrofuran (THF) and protein standards were obtained from Sigma (St. Louis, MO, USA). N-[(3-Perfluorooctyl)propyl]iodoacetamide (FIAM) and fluorous NuTips™ were obtained from Fluorous Technologies Inc. (Pittsburgh, PA, USA). C18 ZipTips™ were obtained from Millipore (Billerica, MA, USA). Tris(2-carboxyethyl)phosphine (TCEP) was obtained from Applied Biosystems (Foster City, CA, USA). The matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) was purchased from Bruker Daltonics (Billerica, MA, USA).
Tryptic digestion of protein standards
Bovine serum albumin (BSA) or holo-myoglobin (100μmg) was dissolved and reduced in 50μL 8M urea, 30 mM NH4HCO3 and 1mM TCEP at 37°C for 1 h, then 40μL ACN (final concentration 10%) and 310μL 100mM NH4HCO3 were added to the solution, followed by the addition of 2 μg of mass spectrometry grade trypsin (Promega, Madison, WI, USA). The digestion was performed overnight at 37°C. Afterwards, the tryptic peptide mixture was acidified with 5% formic acid to terminate the digestion. Desalting of the peptides was performed using an LC-18 SPE column (Supelco, Bellefonte, PA, USA), which was first activated by 3mL ACN and equilibrated by 3mL 0.1% TFA/water solution. After the peptide mixture had been slowly loaded, the column was washed with 2mL of a solution of 0.1% TFA in water. Finally, the peptides were eluted with 1.2mL 50% ACN/0.1% TFA solution, aliquotted, then lyophilized and stored at −20°C before further operations.
Labeling of peptides by FIAM reagent
For the FIAM labeling experiment, 40 nmol of a standard peptide or 0.37 nmol of a BSA tryptic digest were first dissolved in 50 μL 50 mM NH4HCO3/4 M urea with 0.5 mM TCEP and incubated at 37°C for 30 min. Then 200 nmol FIAM dissolved in 50 μL THF was mixed with the peptide solution. The labeling reaction mixture was kept for 30 min at 37°C, and then the FIAM reagent was inactivated by addition of 10 mM DTT. The products were stored at −50°C before further experiments.
Fluorous solid-phase extraction (FSPE)
The FSPE of fluorous-labeled peptides was carried out using three different protocols. Protocol A followed the recommended method of Fluorous Technologies;30
protocols B and C followed the methods optimized by Brittain et al.24
delineates the detailed procedures of each protocol. For each protocol, 25 pmol of labeled Cys-Kemptide was mixed with 25 pmol of a myoglobin digest (without cysteine-containing peptides) and these were used as the starting materials. Purified labeled peptides were lyophilized and dissolved in 25 μL 60% ACN/0.1% TFA for mass spectrometry analysis.
Evaluation of three methods for capture of fluorous-labeled peptides using fluorous NuTips™
Stepwise elution of peptides using ZipTip™ SPE columns
For enrichment of fluorous-labeled peptides using Zip-Tips™, the peptide mixture was dissolved in either 20% or 35% ACN in 0.1% TFA. The ZipTip™ procedure was conducted according to the manufacturer's protocol. Stepwise elution was realized by using steps of increasing concentrations of ACN in 0.1% TFA, as described herein.
MALDI-TOF-MS and peptide identification
The matrix CHCA was freshly reconstituted from re-crystallized stock to 20 μg/μL in 50% ACN/0.1% TFA. A volume of 1 μL of peptide solution was mixed on-target with an equal volume of matrix solution. MALDI-TOF mass spectra were obtained on a Bruker Daltonics Reflex IV™ mass spectrometer operated in positive ion reflectron mode. The intensity of the beam from the nitrogen laser (337 nm, 3 ns pulse width) was adjusted to approximately 6–10% above threshold. External calibration was performed in each case using Bruker Peptide Standards II. For each spectrum, 150 laser shots were summed. The MALDI mass spectra were analyzed with FlexAnalysis 3.0 software (Bruker Daltonics).
LC/ESI-MS/MS analysis was performed using a nanoACQUITY UPLC system (Waters Corp., Framingham, MA, USA) coupled to a Q-TOF-API-US quadrupole orthogonal TOF mass spectrometer (Waters Corp., Milford, MA, USA) equipped with a Z-spray™ source (Waters Corp.). Solution A (1.5% ACN/1% HCOOH) and solution B (98.5% ACN/1% HCOOH) were used for the UPLC system. Peptides were diluted with solution A and injected with an autosampler, pre-concentrated and desalted using an on-line peptide trap column (5 μm, Symmetry™ C18, 180 μm × 20 μm, Waters Corp.). Separation was performed using a high-pressure capillary column (1.7 μm particle size, BEH 130, C18, 150 μm × 100 mm, Waters Corp.) at a flow rate of 0.5 μL/min using the following gradient: 2 min, 3% solution B; 55 min, 55% solution B; 60 min 70% solution B; 62 min 100% B, hold for 8 min. The column was then re-equilibrated with solution A for 20 min before another injection. Mass spectra were acquired in the m/z 400–1700 range under the following conditions: positive polarity, 110°C ion source temperature, 2.1 kV capillary voltage, and 32 V cone voltage. For MS/MS, data-dependent acquisition was programmed as follows: the two most abundant peaks with intensities above 10 counts were selected for MS/MS scans; collision energy was programmed as proportional to the m/z and the charge state of the precursor ion and ranged from 16 to 40 V; two MS/MS spectra were acquired for each precursor ion over the range m/z 100–1800. A 30-s exclusion time window was programmed for previously fragmented parent masses. Data were interpreted with MassLynx software (version 4.1, Waters Corp.).
For direct infusion analyses by ESI-Q-TOF MS, the peptide mixtures were dissolved in 60% ACN/2% HCOOH, and sample were introduced at flow rate of 0.5 μL/min.
MS/MS peak lists were generated by processing LC/MS/MS raw data with ProteinLynx Global Server 2.2.5 using the following parameters: for noise reduction, background subtract type: normal; background threshold: 15%; background polynomial: 5; for deisotoping and centroiding: perform deisotoping: yes; deisotoping type: slow; iterations: 30; threshold: 3%. The fragment ion lists were searched against Swissprot database (version 51.6, 257967 sequences, 93949396 residues) using local MASCOT (Matrix Science) (version 2.2.0) with parameters as follows: species; other mammalian; enzyme, trypsin; variable modifications: fluorous labeling (C), oxidation (M); mass values, monoisotopic; MS and MS/MS tolerance, 0.5 Da, 13C = 1; max missed cleavage, 2; instrument type, ESI-QUAD-TOF.