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Clinical Medicine Insights 

DOI:    https://doi.org/10.52845/CMI/2022-3-2-2 

CMI 03 (02), 297−303 (2022) ISSN (O) 2694-4626

RESEARCH ARTICLE       

Maldi-Tof Analysis of Mitochondrial Peptides 

Adam Good
1
, Alicia Wells

1
, Ben Katz

2
, Michael Alexander

1,3
, Dmytro Klokol

3,4
, 

Mike K.S. Chan4
, Michelle B.F. Wong4

, Desiree C.T. Cox
3
, Jonathan R.T. Lakey

1,4 

Corresponding Author: Jonathan RT Lakey

PhD, MSM, Professor Emeritus University of California Irvine

1
University of California, 

Irvine- Department of 

Surgery, 

2
University of California, 

Irvine- Department of 

Chemistry,  

3
BioPep, 4621 Technology 

Drive, Golden, CO, 80403, 

USA 

4
European Wellness Group, 

Klosterstrasse 205ID, 67480, 

Edenkoben, Germany 

5
University of California, 

Irvine- Department of 

Biomedical Engineering 

Introduction

Peptides are linear polymers formed by a series of 

amino acid residues that are linked together 

through peptide bonds [1]. In comparison to 

proteins, which typically contain between 50 and 

2000 amino acid residues and have a mean 

molecular weight between 5.5 and 22 kDa, 

peptides have fewer than 50 residues and a 

reduced weight. A unique set of proteins and 

peptides are produced in each cell line and these 

play a part in the regulation of biological 

homeostasis [2]. Peptides have been demonstrated 

to play an important role in modulating 

transcription, transmission of biological 

information, and in restoring the genetic 

alterations that occur with age [3, 4]. These 

peptides are signaling molecules that act as 

regulatory factors through their interaction with 

DNA and histone proteins. Moreover, the 

physiological process of aging is highly 

influenced by the peptidergic regulation of 

homeostasis. As part of the aging process, the 

frequency and strength of signals to the 

mitochondria declines, causing signals to be sent 

back to the nucleus that cause the arrest of cell 

proliferation and initiation of apoptosis (Haas, 

2019; Akbari, Kirkwood, and Bohr, 2019) [5-6].  

Abstract 

Short peptides are known to play an important role in modulating 

transcription, in transmitting biological information and in restoring the 

genetic alterations that occur with aging. This paper aims to describe a 

method of identifying the population of peptides within a peptide cocktail 

formulation. A sample of lung-derived Mito Organelle (MO) Peptides 

(LBS) of specific pathogen free (SPF) mammalian rabbits sourced from 

Charles River Labs were analyzed by mass spectrometry and 

chromatograms were generated for further examination. The experimentally 

derived peaks were compared between two batches of LBS MO peptides 

using MALDI-ToF mass spectroscopy. The following report outlines the 

experimental methods and the results from performing MALDI-ToF mass 

spectrometry on various peptides from the company European Wellness 

(EW).  

Keywords: peptides, Mito Organelles (MO) Peptides, mass spectrometry, 

MALDI-ToF 

Copyright : © 2021 The Authors. Published by Medical Editor and 

Educational Research Publishers Ltd. This is an open access article under 

the CC BY-NC-ND license (https://creativecommons.org/lic enses/by-nc-

nd/4.0/). 

https://creativecommons.org/lic%20enses/by-nc-nd/4.0/
https://creativecommons.org/lic%20enses/by-nc-nd/4.0/


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Peptide therapy aims to renew the strength of 

signals received by cells to either induce peptide 

production or renew normal signaling processes, 

thereby rejuvenating and revitalizing tissues as 

well as the organism as a whole [3, 4, 7-9]. 

Although the content of peptides is similar 

between cells, the function and morphology of 

each cell defines the contents of its biologically 

active substances and unique ultrastructures. 

Moreover, certain biologically active substances 

are predominantly synthesized or accumulated in 

specific tissues. Since the signaling activity and 

function of peptides is largely based on the cell 

type, peptide therapy utilizes organ-specific 

extracts to target diseased or aging tissue. Due to 

the short lengths of peptides (<45 residues) and 

their low molecular weights, their biosynthesis 

and extraction processes permit mass production 

and distribution for use in therapeutic treatments 

[3]. Through years of research and extensive 

global practice, MF-Plus has manufactured two 

products, Nano Organo Peptides (NOPs) and Mito 

Organelles (MO) Peptides, which are intended for 

use in both animals and humans as a revitalization 

therapy [10]. 

Mito Organo (MO) peptides are biologically 

extracted mixtures of cellular peptides that have 

predominantly mitochondria-specific functions 

[11]. Although cells of different organ systems 

have similar functions, variations in cellular 

functions between organs creates the differential 

expression of peptides, which can be utilized for 

various therapeutic purposes. MO peptides are 

organ-specific extracts that are aimed at 

revitalizing and rejuvenating mitochondrial 

activity, thereby regenerating cells and organisms 

as a whole [12,13].  

Despite the numerous studies highlighting the 

therapeutic effectiveness of MO peptides, little is 

known of the exact makeup of these formulations. 

Matrix-assisted laser desorption/ionization time of 

flight (MALDI-ToF) mass spectrometry has been 

able to identify and quantify analytes in complex 

solutions and allows for highly sensitive, fast and 

high-throughput analysis [14,15]. In addition, it is 

thought to be able to identify the population of 

peptides derived from peptide cocktail 

formulations. The mass spectrometer produces a 

readout of peaks plotted in relative abundance 

against mass-to-charge ratios. By searching the 

experimentally-derived peaks against a database 

of known proteins, it may be possible to identify 

the peptides. The following report outlines the 

experimental methods and the results of 

performing MALDI-ToF mass spectrometry on 

various EW peptides. 

2. Materials and Methods

Two batches of LBS MO201901 EW peptides 

were suspended in saline solution upon collection. 

Formulations with the characterization LBS were 

extracted from lung samples in specific pathogen 

free (SPF) mammalian rabbits sourced from 

Charles River Labs. All samples were kept on ice 

throughout the duration of the experiment and 

were handled using good laboratory practices.  

Sample Preparation 

The ThermoFisher Scientific BCA Protein Assay 

protocol was utilized to determine the protein 

concentrations of the unknown peptide solution. 

Triplicate sample readings were obtained for the 

peptide solution using the Tecan Infinite F200 

microplate reader at a wavelength of 570 nm.  

Sample Preparation of LBS MO Samples 

Peptides from the LBS MO samples were 

withdrawn at a volume of 30 µg/mL and pipetted 

into auto column tubes in an agarose gel. Sample 

mixtures were separated by molecular weight with 

SDS-PAGE and Coomassie™ blue staining to 

visualize the proteins. An individual protein band 

was cut out from the gel and placed into a low-

binding, siliconized microcentrifuge tube. Proteins 

were then de-stained in the tube with 100μl of a 

1:1 methanol and water solution and vortexed. 

The gel piece was further washed by removing the 

de-stained solution in the tube and adding 400μl 

of water. Tubes were shaken for 15 minutes at 

room temperature, and the de-staining was 

continued until the gel became colorless with a 

minimum of 3 repeats. After destaining, 400μl of 

100% acetonitrile was added to dehydrate the gel 

for 10 minutes and dried by vacuum 

centrifugation after removal of the supernatant. 

Disulfide bonds were removed with the addition 

of 100μl of 10mM dithiothreitol (DTT) to the gel 

and then the gel was incubated for 45 minutes at 

55°C. The solution was removed and 100μl of 

55mM iodoacetamide was added to the gel and 

then incubated for an additional 30 minutes at 



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room temperature under low light conditions, 

allowing trypsin to access cleavage sites. The 

solution was then added to 400μl of the gel and a 

wash solution (50% per volume acetonitrile, 

25mM ammonium bicarbonate) was added. The 

gel was then incubated at room temperature and 

vortexed three times for 15 minutes. The gel was 

subsequently dehydrated with 400μl of 100% 

acetonitrile for 10 minutes and dried in a vacuum 

centrifuge after removal of the supernatant.  

Enzyme Digestion 

A pH 8 protease trypsin solution diluted 1:1000 

with 25mM ammonium bicarbonate was prepared 

and diluted to a final concentration of 10 - 20 

μg/ml. Trypsin was added to the gel and it was 

incubated on ice for 1 hour. The solution was 

removed and replaced with 25mM ammonium 

bicarbonate to cover the gel. The gel was then 

incubated at 37°C overnight. 

Peptide Extraction 

The supernatant containing the peptides was 

transferred to a new microcentrifuge tube. A gel 

extraction solution of 50% per volume 

acetonitrile, 1% trifluoroacetic acid (TFA) and 

49% water was added, which was followed by 

incubation at room temperature and a vortex cycle 

of 20 minutes. This solution was combined with 

the supernatant from the peptides in the new 

microcentrifuge tube.  

Analysis 
10μl of a 1:1 solution of 0.1% TFA and 100% 

acetonitrile was added to 10μl of each sample. 1μl 

of DMP was added onto the MALDI plate. 1μl of 

sample solution was then applied with 1:1 0.1% 

TFA and 100% acetonitrile onto the same spot as 

the DMP and the spot was allowed to dry. The 

samples were run on MALDI in triplicate and the 

peaks were compared against a database to 

identify the key peptides and amino acids found in 

the samples.  

Data Analysis 

Data was analyzed using the chromatogram tool in 

MassLynx software. Replicates for each batch 

were analyzed together and chromatograms for 

each were generated. The masses of the three most 

prominent peaks were deconvoluted and the key 

peptides and amino acid components were 

determined against open-source databases.  

3. Results

The concentration of peptides in each batch was 

initially determined via the ThermoFischer™ 

Scientific BCA Protein Assay (Figure 1). The 

average protein concentration of 252.0 μg/mL 

( 12.83μg/mL) and 260.6μg/mL ( 32.23μg/mL) 

was determined for Batch 1 and Batch 2 

respectively. There were no statistically 

significant differences between batches (p = ns).

Figure 1. Average protein concentration (ug/mL) of Batch 1 and 2 LBS MO201901 peptides.  

Standard error bars are included. No statistically significant differences were found between batches (p=ns). 

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Jonathan RT Lakey et al.



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The samples were then separated by molecular 

weight with SDS-PAGE and digested with the 

protease trypsin. Once prepared, the samples were 

run on MALDI-Tof in triplicate and 

chromatograms were generated (Figure 2). The 

deconvoluted masses of Batch 1 (Figure 3) and 

Batch 2 (Figure 4) of LBS MO show similarity in 

major protein components. The deconvoluted 

masses in Batch 1 represent five major 

components with sizes of 14,969 Da, 15,300 Da, 

8,449 Da, 8,294 Da and 4,618 Da respectively. 

Batch 2 results showed six major protein masses 

of 14,969 Da, 15,301 Da, 8,294 Da, 8,449 Da, 

5,436 Da, and 6,214 Da respectively. 

Figure 2. Chromatograms obtained from Batch 1 (A) and Batch 2 (B) of LBS MO201901. 

Red arrows pointing upwards on the x-axis of 

each chromatogram identify peaks that correspond 

to differing peptide components in the solution. 

The green, blue, and red chromatograms 

associated with both Batch 1 and Batch 2 illustrate 

different runs of the sample. 

Figure 3. Deconvoluted mass spectrometry data obtained from LBS MO Batch 1. 

Five peptide fragments are ordered from left to 

right in decreasing order of abundance within the 

sample. The relative sizes of peptides from left to 

right are 14,969 Da, 15,300 Da, 8,449 Da, 8,294 

Da and 4,618 Da respectively. 



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Figure 4. Deconvoluted mass spectrometry data obtained from LBS MO Batch 2. 

Six peaks representing six major peptide 

fragments are ordered from left to right in 

decreasing order of abundance within the sample. 

The relative sizes of peptides from left to right are 

14,969 Da, 15,301 Da, 8,294 Da, 8,449 Da, 5,436 

Da, and 6,214 Da respectively. 

4. Discussion

Short peptides are known to play a critical role in 

modulating the transmission and transcription of 

biological information and have been shown to 

decline throughout the natural process of aging 

[2]. Peptide therapy aims to either reinstate normal 

signaling patterns by renewing the strength of the 

signals received by cells or induce the cells to 

begin to produce peptides of their own [16]. Since 

different tissue types produce different peptides, 

peptide therapy utilizes organ-specific extracts to 

target aging or diseased tissue with the goal of 

revitalizing normal peptidergic signaling in these 

regions and improving overall health and 

wellbeing [17].  

Through years of testing and development, 

European Wellness (EW) and MF-Plus have 

manufactured Mito Organelles (MO) peptides that 

are intended for use in peptide therapy in both 

animals and humans [18]. Despite numerous 

studies demonstrating the potential of MOs in 

therapeutic applications such as cosmetics [19] 

and regenerative organ repair [11], little research 

has been done to investigate and identify the key 

peptides in these solutions. Mass spectrometry 

(MS) is a chemical analysis technique that enables 

the direct identification of molecules based on 

their mass-to-charge ratios and fragmentation 

patterns [20]. By comparing experimental MS 

data with that of well-established open-source 

databases, the identity of the molecules, peptides, 

or proteins can be found within a solution. Due to 

the low-cost and rapid application of MS in 

identifying the components of unknown solutions, 

our study employed MS as our primary method of 

identification.  

MALDI-TOF utilizes a protein fingerprinting 

method in which the sample is digested by a 

proteolytic enzyme such as trypsin and used to 

generate an MS spectrum that can be searched 

against existing databases [21, 22]. Matched hits 

are ranked according to a scoring method in which 

the candidate protein that contains more 

proteolytic peptides has a higher score and 

generally represents the most probable 

protein/peptide. The desirability of MALDI-TOF 

also includes the speed at which each run is 

performed– often less than one minute to obtain– 

and the speed at which analysis can be performed 

against a database.  

Conclusion: 

The results of our study using MALDI-TOF to 

analyze the MO LBS sample indicated that there 

were five major peptide products of interest in 

Batch 1 and six major peptide products in Batch 2 

(Figure 3 and 4). Following deconvolution, five 

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Jonathan RT Lakey et al.



CMI 03 (02), 297−303 
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peptides were identified in Batch 1 (Figure 3) with 

masses of 14,969 Da, 15,300 Da, 8,449 Da, 8,294 

Da and 4,618 Da. Batch 2 identified four of the 

same peptides– 14,969 Da, 15,301 Da, 8,294 Da, 

8,449 Da in size– and two additional peptides of 

5,436 Da, and 6,214 Da in size (Figure 4). Slight 

differences in peptide products between batches is 

likely due to the heterogeneous nature of 

cellularly-derived solutions and differences that 

occurred during the extraction process. Further 

research must be conducted to confirm the identity 

of the peptides and discover their significance in 

peptide therapy. 

Supplementary Materials: The following 

supporting information can be downloaded at: 

www.mdpi.com/xxx/s1, Figure S1: title; Table S1: 

title; Video S1: title. 

Author Contributions: Conceptualization, J.L., 

M.W., M.C., D.K., D.C.; methodology, B.K., 

M.A.; validation, B.K.; formal analysis, B.K.; 

investigation, A.W., A.G., B.K.; data curation, 

A.W., A.G., B.K.; writing—original draft 

preparation, A.W., A.G.; writing—review and 

editing, A.W., A.G., J.L.; visualization, J.L., 

M.W., M.C., D.K.; supervision, J.L., M.W., M.C., 

D.K., D.C., B.K., A.M.; project administration, 

J.L., M.W., M.C., D.K., D.C., B.K.; funding 

acquisition, J.L., M.W., M.C., D.K., D.C.. All 

authors have read and agreed to the published 

version of the manuscript. 

Funding: This research was funded by a grant to 

the University of California, Irvine from European 

Wellness Biomedical Group.   

Institutional Review Board Statement: Not 

applicable 

Data Availability Statement: The data presented in 

this study are available in the study outlined. 

Acknowledgments: The authors wish to 

acknowledge the support of the Department of 

Surgery and the Core Mass Spectrometry 

laboratory of the University of California, Irvine 

for their support.  

Conflicts of Interest: The funders had no role in 

the design of the study; in the collection, analyses, 

or interpretation of data; in the writing of the 

manuscript, or in the decision to publish the 

results. 

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