TECHNOTE:
Sample Preparation for Proteomics
Applications with PIXUL™

Cells

TissueProteins

TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM
Introduction

HEK293T Cells

Sample preparation in Proteomics studies ischallenging due to the complexity of the proteome.
The first and often most critical step in the sample
preparation protocol is cell lysis which is carried out
using sonication. Due to its accuracy, sensitivity, andflexibility Mass Spectrometry has become the gold
standard for analyzing the proteome. In recent years
it has also found its way into the clinical setting.

Sample Preparation

In this scenario reproducibility and the ability forhigh-throughput processing is crucial. With Active
Motif’s PIXUL™ Multi-Sample Sonicator it is possible
to shear 96 samples with 12 different settings in ahigh-throughput fashion, which can be applied tomany different sample types. With this TechNote,
we want to demonstrate this flexibility by comparing
the lysis of HEK293T cells, and mouse liver tissue
samples with the PIXUL Multi-Sample Sonicator to a
standard probe sonicator.
After cell lysis, samples were subjected to LC-MS/
MS analysis on an LTQ Orbitrap Elite instrument.
Comparison of both sonication methods was
performed by evaluating common QC parameters,
including the protein extraction efficiency, the numberof identified proteins, and the presence of unwanted
protein modifications.

Sample preparation for MS-Analysis was performed
using the Protifi S-Trap™ sample processing
technology. In brief: A cell pellet containing 5.4M
HEK293T cells was resuspended in a buffer
containing 5 % SDS in 50 mM TEAB pH 8.5. Cell lysis
was enhanced via Sonication. The standard approach
is lysis using a probe sonicator (VCX130 with a 3 mmprobe, Sonics and Materials, Newtown, USA) with
3x15 s pulses, 15 W output, and 30 s on ice between
individual bursts.
This approach was compared to the PIXUL MultiSample Sonicator with the following settings: 5 minof shearing, 50 pulses of 2 MHz with a frequency of 1
kHz, 20 Hz burst rate, and 2 wells per sample whichwere pooled later in the process.
After sonication, DTT and IAA were added to reduceand alkylate the proteins and proteins were digested
with 1 µg Trypsin at 37 °C o/n. 1.5 µg of the resulting
peptide mixture was then analyzed by nanoLCMS/MS on an LTQ Orbitrap Elite instrument using
standard 2 hour runs and CID fragmentation.
Protein Concentration
The first QC measure taken in the protocol was todetermine whether the protein yield was comparable
between the standard probe sonicator and the PIXUL.
To evaluate protein concentration, samples were
subjected to BCA analysis and analyzed in triplicate.

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TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM

Figure 1: Protein Concentration Measurement with BCA

Three samples processed with the standard
probe sonicator or the PIXUL were subjected to
BCA analysis and the protein concentration isshown in µg/ml. Orange: Samples processed with
PIXUL. Green: samples processed with the probesonicator.

Protein concentration of samples processed with
PIXUL are in the same range (4890 - 6543 µg/ml) as
samples processed in the standard way (4247 - 4761
µg/ml), indicating a similar, if not slightly better protein
yield when using PIXUL compared to the standard
method.

Figure 1 shows a comparison between threesamples processed with the standard probesonicator and three separate samples processedwith PIXUL.

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TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM

Table 1: Comparison of the Number of identified Peptides and Proteins.

Number of Identified Peptides
Despite small differences in the extracted protein
concentration, for each sample the same peptide
amount was injected for LC-MS/MS analysis.
LC-MS/MS runs of all 6 samples were searchedseparately using the MaxQuant algorithm (version
1.6.17.0) with mainly default settings, including a
false discovery rate set at 1% on both peptide andprotein level.

The top three rows represent PIXUL data and thelower three rows the standard probe sonicator.
The number of peptides and proteins that wereidentified was comparable between both groups(PIXUL: 9080-10020, Standard: 9559-9619), indicating
a similar ability to identify peptides and proteinsin HEK293T cell lysates. The variability between
replicates, however, is slightly higher with PIXUL.

Spectra were searched against the human
Swiss-Prot Reference proteome database(www.uniprot.org) (database release version of
2021_01, containing 20,621 protein sequences).

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TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM

Figure 2: Average percentage of missed cleavages.

Missed Cleavages
Next, the number of missed trypsin cleavages
was evaluated. Missed cleavages represent an
instance where Trypsin did not cleave the peptide
as expected after each Lysine or Arginine residue.
During database searching, up to 2 missedcleavages were allowed as a standard setting,hence the number of peptides with 0, 1, or 2 missedcleavage events could be assessed.
The average percentage of missed cleavages isshown for PIXUL (orange) and standard Probe
Sonicator (green). The total percentage of missedcleavages is comparable between PIXUL andthe standard probe sonicator (33,79 vs 33,35).
Furthermore, the fraction of 0, 1 or 2 missed cleavage
events is comparable, suggesting a similar degree of
missed cleavages in samples prepared with PIXUL
compared to the standard probe sonicator.

Overall, the number of missed cleavages is
acceptable given the fact that no (e.g., endoLys)
pre-digestion was performed.
Modifications
Last, the average percentages of Acetylated
Protein N-termini and Oxidized Methionine
residues (methionine sulfoxide) were assessed
as measures for the ability to detect protein
modifications and as general quality parameters
for sample preparation including sonication.

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TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM

Figure 3: Average percentage of protein N-terminal acetylation and oxidized methionine.

LEFT: The average percentage of protein
N-terminal acetylation is shown for PIXUL (orange)and standard probe sonicator (green).
RIGHT: The average percentage of oxidized
methionine is shown for PIXUL (orange) andstandard Probe Sonicator (green).

Altogether, these results showed that for Proteomics
sample preparation of standard culture cells, the
PIXUL gives comparable results to standard Probe
sample preparation protocol with the benefit ofhigher throughput.

The percentage of acetylated protein N-termini insamples processed with PIXUL (2.7 %) as well as thepercentage of oxidized methionines (11.72 %) was in
the same range as the percentage observed withthe standard probe sonicator (2.47 %, 11.09 %).

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TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM
Mouse Liver Tissue
Sample Preparation
Sample preparation for MS-Analysis was performed
using the Profiti S-Trap™ sample processing
technology. In brief: 100 mg of fresh mouse livertissue was resuspended in a buffer containing 5 %SDS in 50 mM TEAB pH 8.5. Tissue disruption was
performed using a MACS tissue dissociator (Miltenyi
Biotech).
The standard approach then includes sonication of
the sample using a probe sonicator (VCX130 with a
3 mm probe, Sonics and Materials, Newtown, USA)with 3x15 s pulses, 15 W output and 30 s on ice
between individual bursts.

This approach was compared to the PIXUL MultiSample Sonicator with the following settings: 5, 15,30 or 60 min of shearing, 50 N pulse with 1 kHz, 20
Hz burst rate and 8 wells per sample which werepooled later in the process.
After sonication DTT and IAA were added toreduce and alkylate the proteins and proteins weredigested with 1 µg Trypsin at 37°C o/n. 1.5 µg of theresulting peptide mixture was then analyzed bynanoLC-MS/MS on an LTQ Orbitrap Elite instrument
using standard 2 hour runs and CID fragmentation.

Figure 4: The samples processed with the standard probe sonicator or the PIXUL were subjected to
BCA analysis and the protein concentration is shown in µg/ml.
Orange: Samples processed with PIXUL, green: samples processed with the probe sonicator,
blue: samples selected for further analysis.
P-value5min=0.00048

P-value15min=0.00552

P-value30min=0.014

P-value60min=0.022

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TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM
Protein Concentration
The first QC measure taken in the protocol
was, whether the Protein yield was comparable
between the standard Probe Sonicator and thedifferent durations used on the PIXUL. To evaluate
Protein concentration, samples were subjected to
BCA analysis and analyzed in triplicate.
Figure 4 shows a comparison between six samples
processed with the standard probe sonicator and8 samples processed in duplicates for 4 different
time points (5 min, 15 min, 30 min and 60 min)with PIXUL.

Protein concentration of samples processed with
PIXUL are in the same range (1409 - 2710 µg/ml)
as samples processed in the standard way (1449 1764 µg/ml), indicating a similar, if not slightly better
protein yield when using PIXUL compared to thestandard method. In addition, there was no obvious
difference between the samples sheared for varying
amounts of time on the PIXUL, indicating that 5
min of shearing is already sufficient to yield protein
concentrations sufficient for downstream analysis.

Table 2: Comparison of the Number of identified Peptides and Proteins.
Top eight rows represent data of two replicates of each of the four durations run on PIXUL andlower two rows represent the standard probe sonicator.

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TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM
Number of Identified Peptides
The first QC measure taken in the protocol was,whether the Protein yield was comparable between
the standard Probe Sonicator and the different
durations used on the PIXUL. To evaluate Protein
concentration, samples were subjected to BCA
analysis and analyzed in triplicate.
Despite small differences in the extracted protein
concentration, for each sample the same peptide
amount was injected for LC-MS/MS analysis.

LC-MS/MS runs of selected samples were searchedseparately using the MaxQuant algorithm (version
1.6.17.0 ) with mainly default settings, including a false
discovery rate set at 1% on both peptide, and protein
level. Spectra were searched against the human
Swiss-Prot Reference proteome database(www.uniprot.org) (database release version of
2021_01, containing 20,621 protein sequences).

Figure 5: Average percentage of missed cleavages.
The average percentage of missed cleavages is shown for PIXUL (orange, purple, black, grey)
and Probe sonicator (green).

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TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM
The number of peptides and proteins that wereidentified were comparable between bothgroups (PIXUL: 7265-8380, Standard: 7803-8124)
indicating a similar ability to identify peptides
and proteins in mouse liver tissue. Furthermore,
the ability to identify peptides did not vary
substantially between samples using different
processing times on the PIXUL, meaning that 5
minutes of sonication is sufficient to completely
and efficiently lyse liver tissue.

Missed Cleavages
Next, the number of missed trypsin cleavages wasevaluated. Missed cleavages represent instanceswhere trypsin did not cleave the peptide as
expected after each Lysine or Arginine residue.
During database searching up to 2 missedcleavages were allowed as a standard setting,hence the number of peptides with 0, 1 or 2 missedcleavage events could be assessed.

Figure 6: Average percentage of Protein N-terminal acetylation and oxidized methionine.
LEFT: The average percentage of Protein N-terminal acetylation is shown for PIXUL (orange) and standard
Probe Sonicator (green). RIGHT: The average percentage of oxidized methionine is shownfor PIXUL (orange) and standard Probe Sonicator (green).

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TECHNOTE:
Sample Preparation for Proteomics Applications with PIXULTM
The total percentage of missed cleavages iscomparable between PIXUL and the standard
probe sonicator (30,74-35,72 vs 36,05).
Furthermore, the fraction of 0, 1 or 2 missedcleavage events is comparable, suggesting asimilar degree of missed cleavages in samples
prepared with PIXUL compared to the standard
probe sonicator. Moreover, the fraction of 0, 1 or 2
missed cleavages among the varying processing
time on PIXUL is comparable. Overall, the numberof missed cleavages is acceptable given thefact that no (e.g., endoLys) pre-digestion was
performed.
Modifications
Last, the average percentages of acetylated
protein N-termini and oxidized Methionine residues(methionine sulfoxide) were assessed as measures
for the ability to detect protein modifications and as
general quality parameters for sample preparation
including sonication.
The percentage of acetylated protein N-termini aswell as the percentage of oxidized methionine insamples processed with PIXUL was in the samerange as samples processed with the standard
probe sonicator.

Altogether, these results showed that for Proteomics
sample preparation of mouse liver tissue as arepresentation for animal tissue samples, the PIXULMulti-Sample Sonicator gives comparable results
to the probe sample preparation protocol with theadded benefit of higher throughput.

Summary
With this short TechNote, we demonstrate thesuitability of PIXUL Multi-Sample Sonicator forProteomics preparation of HEK293T cells andmouse liver tissue samples using the S-Trap
System. Results were compared to the standard
way of processing those samples using a probe
tip sonicator.
To evaluate the suitability of the samples for
LC-MS/MS analysis, the extracted protein
concentration, the number of identified peptides
and proteins, the percentage of missed trypsin
cleavages and the presence of common protein
modifications was measured. For all evaluatedsample types and quality parameters, the PIXUL
performed equally well or better as the standard
probe tip sonicator, with the benefit of being able
to process 96 samples in a short period of time.
The reduced hands-on time further enables thedevelopment of semi-automated workflows forProteomics sample preparation.

Data courtesy of the VIB Proteomics Core.

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