لخّصلي

2.1.Proteome Discoverer, which is a software trademark belonging to Thermo Fisher Scientific, provides access to annotation information from ProteinCenter, a web-based application used to retrieve biologically enriched data for individual proteins.Chemicals Water, methanol, acetonitrile (ACN), trifluoroacetic acid (TFA) and formic acid (FA), hexane, chloroform, Tris-HCl, ammonium bicarbonate (ABC), dithiothreitol (DDT), iodoacetamide (IAA), 2-cloroacetamide (CAA), sodium deoxycholate (SDC), trypsin, bovine serum albumin (BSA), C18 resin, nitric acid and Coomassie brilliant blue G-250 dye were purchased from Merck (Milan, Italy).Acquisitions in full scan mode were performed in the range of m/z 350-1600 with a resolution power of 120k using an automatic gain control (AGC) target of 3 x 106 and an injection time (IT) of 100 ms. Full-MS/ddMS2 acquisitions were conducted using normalized collision energy (NCE) fixed at 28 with a resolution of 15k, AGC of 1 x 105 , IT of 100 ms, isolation window of 1.2 m/z, minimum AGC of 1 x 103 and dynamic exclusion of 30 s. Instrumentation control is achieved using Xcalibur software 2.2 SP1.48 (Thermo Scientific).In solution digestion Protein digestion was performed starting from dried protein extracts obtained using Protocols I and II. The procedure began by adding 20 uL of 50 mM ABC and 2 uL of 100 mM dithiothreitol (DTT) to achieve a final DTT concentration of 10 mM. The mixture was incubated at 56 oC with shaking at 400 rpm for 30 min to reduce disulfide bonds.40 uL of 0.1 % TFA and 1 uL of resin C18 were ad alysis of extracted and purified protein digest samples was performed with an EASY-nLC(TM) 1200 nHPLC chromatograph (Thermo Fisher Scientific, USA) coupled with a Q-Exactive mass spectrometer (Thermo Scientific, USA) through an ESI interface.In the processing workflow, LC-MS conditions were set, including m/z tolerance (5 ppm for precursor mass, 0.05 Da for fragment mass), mass range (350-7000 Da for precursor mass), fragmentation type (higher-energy collisional dissociation, HCD), protease (trypsin), constant modification (carbamidomethylation of cysteine), and dynamic modification (oxidation of methionine).Bolt sample buffer, Nupage Bis-Tris gel 4-12 %, MES buffer, Instant Blue, prestained protein ladder, Bolt reducing agent and tris(2-carboxyethyl) phosphine (TCEP) were purchased from Thermo Fisher Scientific (Waltham, US).The supernatant was removed, and the protein pellet was dried under a nitrogen flow and stored at - 20 oC. Protocol III: Building upon a similar detergent combination described by Deb-Choudhury et al. (2016), this protocol was developed to enhance protein extraction efficiency through a synergistic interplay of key reagents (Deb-Choudhury et al., 2016).Proteins were then separated using precast NuPAGE(TM) Bis-Tris Plus gels (4-12 % gradient polyacrylamide) in MES SDS Running Buffer (Thermo Fisher Scientific), which provides optimal resolution for low to medium molecular weight proteins. Together, these reagents significantly boost protein yield and ensure a comprehensive extraction of proteins from diverse cellular compartments, providing a robust foundation for downstream analyses (Danko et al., 2022).Protocol I (Bianco, Calvano, et al., 2022): 9.5 mL of 50 mM Tris-HCl buffer was added to a small aliquot of powdered algae sample (5-8 mg of Spirulina and Chlorella in three replicates).The separation was performed at 20 oC using Dr Maisch precolumn (3.5 cm, 100 um ID precolumn of Reprosil-Pur 120 C18-AQ, 5 um) coupled to a Dr Maisch analytical column (18 cm, 75 um ID analytical column of Reprosil-Pur 120 C18-AQ, 3 um), using water (solvent A) and acetonitrile (solvent B) both containing 0.1 % v/v formic acid.In detail, the gradient used during each chromatographic run, at a flow rate of 250 nL/min, was the following: 0-3 min linear from 1 to 3 % (v/v) of B, 3-58 min linear from 3 to 35 % (v/v) of B, 58-63 min linear from 35 to 45 % (v/v) of B, 63-66 min from 45 to 100 % (v/v) of B, 66-74 at 100 % of B. The MS parameters were the following: spray voltage, 2.0 kV (positive polarity); capillary temperatures, 275 oC; S-Lens RF Level, 50 (arbitrary units).SDS-PAGE For electrophoresis, each protein extract was resuspended in a sample buffer consisting of 2 % (w/v) SDS, 40 % (v/v) glycerol, 0.02 % (w/v) bromophenol blue, 0.08 M Tris-HCl (pH 8.0), and 10 % Bolt(TM) Sample Reducing Agent.A prestained protein standard was included as a molecular weight marker, and gels were stained with colloidal Coomassie G-250 for protein visualization, following established protocols (Arenas et al., 2021).So, the organic phase was carefully removed and discarded, and the aqueous phase, containing the protein component, was divided into 0.1 mL aliquots, which were dried under a nitrogen flow and preserved.The reducing agent TCEP and the alkylating agent IAA specifically target disulfide bonds, promoting effective protein denaturation while preventing aggregation.Protein quantification The total protein content in the samples was quantified using the Pierce(R) Microplate BCA Protein Assay Kit - Reducing Agent Compatible (Bainor et al., 2011).Pierce Microplate BCA Protein Assay Kit - Reducing Agent Compatible were purchased by Thermo Fisher Scientific.Meanwhile, the detergent SDC plays a pivotal role in disrupting the lipid bilayer, facilitating the solubilization of membrane proteins.Mass spectra processing was conducted using SigmaPlot 14.5.2.2.2.3.2.4.2.5.2.6.2.8.

2.1. Chemicals
Water, methanol, acetonitrile (ACN), trifluoroacetic acid (TFA) and
formic acid (FA), hexane, chloroform, Tris-HCl, ammonium bicarbonate
(ABC), dithiothreitol (DDT), iodoacetamide (IAA), 2-cloroacetamide
(CAA), sodium deoxycholate (SDC), trypsin, bovine serum albumin
(BSA), C18 resin, nitric acid and Coomassie brilliant blue G-250 dye
were purchased from Merck (Milan, Italy). Bolt sample buffer, Nupage
Bis-Tris gel 4–12 %, MES buffer, Instant Blue, prestained protein ladder,
Bolt reducing agent and tris(2-carboxyethyl) phosphine (TCEP) were
purchased from Thermo Fisher Scientific (Waltham, US). Pierce Microplate BCA Protein Assay Kit - Reducing Agent Compatible were purchased by Thermo Fisher Scientific. The A. platensis and C. vulgaris
microalgae were bought in capsules from LongLife nutritional
supplements.
2.2. Protein extraction protocols
Three different extraction protocols were investigated and, after
testing different conditions, the chosen ones are reported here.
Protocol I (Bianco, Calvano, et al., 2022): 9.5 mL of 50 mM Tris-HCl
buffer was added to a small aliquot of powdered algae sample (5–8 mg of
Spirulina and Chlorella in three replicates). The solution was vortexed
and incubated at 55 ◦C for 1 h at 400 rpm and once the solution reached
room temperature (RT), 0.5 mL of methanol was added. The suspension
was vortexed and sonicated with a probe sonicator for 5 cycles (20 s 50
% power – 20 s break), to facilitate the extraction of proteins. To remove
the lipid component, 3.5 mL of hexane was added, and the solution was
vortexed and centrifuged at RT (15 min at 5000 g). So, the organic phase
was carefully removed and discarded, and the aqueous phase, containing the protein component, was divided into 0.1 mL aliquots, which
were dried under a nitrogen flow and preserved.
Protocol II (Vertommen et al., 2010; Bianco et al., 2020a, b): This
protocol uses a chloroform/methanol solution for the precipitation of
proteins. The algae solutions were prepared by mixing a small aliquot of
powdered algae sample (3–4 mg of Spirulina and Chlorella) with 2 mL of
water. The solutions were sonicated with the probe for 5 cycles (20 s 50
% power – 20 s break) to completely suspend the microalgae samples.
Then, 4 mL of methanol was added. After the vortex, 1 mL of chloroform
and 3 mL of water were added, and the solution was vigorously mixed
and centrifuged at RT (10 min at 9000 g). The aqueous phase was
removed, and 4 mL of methanol was added to the organic phase. Subsequently, the solution was vortexed and centrifuged again at RT (30
min at 9000 g). The supernatant was removed, and the protein pellet was
dried under a nitrogen flow and stored at − 20 ◦C.
Protocol III: Building upon a similar detergent combination
described by Deb-Choudhury et al. (2016), this protocol was developed
to enhance protein extraction efficiency through a synergistic interplay
of key reagents (Deb-Choudhury et al., 2016). The reducing agent TCEP
and the alkylating agent IAA specifically target disulfide bonds, promoting effective protein denaturation while preventing aggregation.
Meanwhile, the detergent SDC plays a pivotal role in disrupting the lipid
bilayer, facilitating the solubilization of membrane proteins. This action
enables the extraction of a wider range of hydrophobic proteins,
including both cytosolic and membrane-associated species. Together,
these reagents significantly boost protein yield and ensure a comprehensive extraction of proteins from diverse cellular compartments,
providing a robust foundation for downstream analyses (Danko et al.,
2022).
A 50 mL solution of OPB buffer contains 10 mM TCEP, 40 mM CAA,
100 mM ABC, 1 % SDC, and pH ± 8.5. First, all the components except
for the SDC were mixed. The pH was measured and adjusted to 8.5 if
needed (for 50 mL of solution, use ca. 60 μL of NH4OH 25 %). The SDC
was added, and the solution was stirred without producing many
L.S. Guadalupi et al. LWT 222 (2025) 117647
2
bubbles. If it was not dissolved or formed gel blobs, it was warmed at 56 ◦C until completely dissolved and liquified.
A buffer OPB (1.0 mL) was added to a small aliquot of micro-algae
(3–4 mg), and the solution was resuspended gently pipetting up and
down with buffer OPB. Then, a probe sonicator was used for 5 cycles (20
s 50 % power – 20 s break). Each sample was divided into two equal
parts. One of these was centrifuged (10 min at 9000 g) to split the pellet
from the supernatant. For each sample, three fractions were obtained,
named whole, pellet, and supernatant. They were incubated at 80 ◦C for
10 min and stored in ice.
2.3. Protein quantification
The total protein content in the samples was quantified using the
Pierce® Microplate BCA Protein Assay Kit – Reducing Agent Compatible
(Bainor et al., 2011). In each microplate well, 9 μL of various solutions
were added: replicate standard controls (prepared with 200 μL of the
same buffer as the unknown samples but without a reducing agent),
sample controls (prepared similarly but with a reducing agent), standards (four solutions of BSA prepared in the same buffer as the samples),
and the unknown samples. Then, 4 μL of compatibility reagent solution
was added to each well. The plate was covered, shaken for 1 min on a
plate shaker, and incubated at 37 ◦C for 15 min. Next, 260 μL of working
reagent containing BCA was added to each well. The plate was again
covered, mixed on the plate shaker for 1 min, and incubated at 37 ◦C for
an additional 30 min. Finally, absorbance readings were measured at
562 nm using a microplate reader.
2.4. SDS-PAGE
For electrophoresis, each protein extract was resuspended in a
sample buffer consisting of 2 % (w/v) SDS, 40 % (v/v) glycerol, 0.02 %
(w/v) bromophenol blue, 0.08 M Tris-HCl (pH 8.0), and 10 % Bolt™
Sample Reducing Agent. The samples were heated at 70 ◦C for 10 min to
ensure complete denaturation. Proteins were then separated using precast NuPAGE™ Bis-Tris Plus gels (4–12 % gradient polyacrylamide) in
MES SDS Running Buffer (Thermo Fisher Scientific), which provides
optimal resolution for low to medium molecular weight proteins. A prestained protein standard was included as a molecular weight marker,
and gels were stained with colloidal Coomassie G-250 for protein visualization, following established protocols (Arenas et al., 2021).
2.5. In solution digestion
Protein digestion was performed starting from dried protein extracts
obtained using Protocols I and II. The procedure began by adding 20 μL
of 50 mM ABC and 2 μL of 100 mM dithiothreitol (DTT) to achieve a final
DTT concentration of 10 mM. The mixture was incubated at 56 ◦C with
shaking at 400 rpm for 30 min to reduce disulfide bonds. Following this,
iodoacetamide (IAA) was added to reach a final concentration of 30 mM,
and the solution was incubated in the dark at 25 ◦C with shaking at 400
rpm for 30 min to alkylate the reduced thiols. Subsequently, DTT was
reintroduced to a final concentration of 5 mM to quench excess IAA. The
solution was then diluted with 100 μL of 50 mM ABC. Proteins were
digested overnight at 37 ◦C with trypsin at a 1:20 enzyme-to-protein
ratio (w/w) while shaking at 400 rpm. The reaction was acidified to
pH 2 by adding 2 % trifluoroacetic acid (TFA; approximately 25 μL) to
terminate the proteolytic activity. For Protocol III, tryptic digestion was
performed directly in the extraction solution without prior drying.
Trypsin was added at a 1:20 enzyme-to-protein ratio (w/w), and the
reaction proceeded overnight at 37 ◦C. The digestion was similarly
terminated by acidifying the solution with 2 % TFA.
2.6. Micro purification for LC-MS
A column was packed in a p10 tip with a small piece of C18 disk. 40
μL of 0.1 % TFA and 1 μL of resin C18 were ad
alysis of extracted and purified protein digest
samples was performed with an EASY-nLC™ 1200 nHPLC chromatograph (Thermo Fisher Scientific, USA) coupled with a Q-Exactive mass
spectrometer (Thermo Scientific, USA) through an ESI interface. The
separation was performed at 20 ◦C using Dr Maisch precolumn (3.5 cm,
100 μm ID precolumn of Reprosil-Pur 120 C18-AQ, 5 μm) coupled to a
Dr Maisch analytical column (18 cm, 75 μm ID analytical column of
Reprosil-Pur 120 C18-AQ, 3 μm), using water (solvent A) and acetonitrile (solvent B) both containing 0.1 % v/v formic acid. In detail, the
gradient used during each chromatographic run, at a flow rate of 250
nL/min, was the following: 0–3 min linear from 1 to 3 % (v/v) of B, 3–58
min linear from 3 to 35 % (v/v) of B, 58–63 min linear from 35 to 45 %
(v/v) of B, 63–66 min from 45 to 100 % (v/v) of B, 66–74 at 100 % of B.
The MS parameters were the following: spray voltage, 2.0 kV (positive polarity); capillary temperatures, 275 ◦C; S-Lens RF Level, 50
(arbitrary units). Acquisitions in full scan mode were performed in the
range of m/z 350–1600 with a resolution power of 120k using an
automatic gain control (AGC) target of 3 × 106 and an injection time (IT)
of 100 ms. Full-MS/ddMS2 acquisitions were conducted using normalized collision energy (NCE) fixed at 28 with a resolution of 15k, AGC of
1 × 105
, IT of 100 ms, isolation window of 1.2 m/z, minimum AGC of 1
× 103 and dynamic exclusion of 30 s. Instrumentation control is achieved using Xcalibur software 2.2 SP1.48 (Thermo Scientific). Mass
spectra processing was conducted using SigmaPlot 14.5. Proteome
Discoverer (version 2.4, Thermo Fisher Scientific) was used to process
the spectra obtained in Full MS/ddMS2 mode.
2.8. Data processing
Each tryptic digest was analyzed using RP-nLC-ESI-FTMS/MS in
data-dependent mode (ddMS2
). The raw data were processed using
Proteome Discoverer™; two specific workflows were created for this
purpose. In the processing workflow, LC-MS conditions were set,
including m/z tolerance (5 ppm for precursor mass, 0.05 Da for fragment
mass), mass range (350–7000 Da for precursor mass), fragmentation
type (higher-energy collisional dissociation, HCD), protease (trypsin),
constant modification (carbamidomethylation of cysteine), and dynamic
modification (oxidation of methionine). For the qualitative analysis,
peptide confidence was set to high, with a minimum peptide length of 4.
Reference database A. platensis for Spirulina and Chlorophyta for
Chlorella microalgae were used while a specific database for contaminants to avoid interference was also implemented. Protein analysis parameters required a minimum of one peptide sequence. Proteome
Discoverer, which is a software trademark belonging to Thermo Fisher
Scientific, provides access to annotation information from ProteinCenter, a web-based application used to retrieve biologically enriched
data for individual proteins. Through this p