Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 11, pp. 1521-1535 © The Author(s) 2025. This article is an open access publication.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 11, pp. 1621-1637.

1521

REVIEW

Methodological Toolbox

for Identifying and Studying Micropeptides:

From Genome to Function

Aleksandr I. Lavrov

1,2

, Nikita M. Shepelev

1,3

, Olga A. Dontsova

1,3

and Maria P. Rubtsova

1,3,a

Lomonosov Moscow State University, Faculty of Chemistry,

119991 Moscow, Russia

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119992 Moscow, Russia

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,

117997 Moscow, Russia

e-mail: mprubtsova@gmail.com

Received July 22, 2025

Revised October 2, 2025

Accepted October 6, 2025

Abstract— Micropeptides encoded by small open reading frames (sORFs) represent a novel, actively studied

class of functional molecules regulating key cellular processes. Studying micropeptides is complicated by

methodological challenges, in particular, their small size, low cellular abundance, and difficulty in generating

specific antibodies. The review systematizes modern approaches to the identification and functional character-

ization of micropeptides. The main strategies for their discovery include the use of bioinformatic algorithms,

global translation analysis via ribosome profiling, direct detection using mass spectrometry-based proteomics,

and phenotypic screenings. The methods for confirming the functions of micropeptides and elucidating mo-

lecular mechanisms of their action genetic knockouts, affinity tagging for visualization, and investigation of

protein-protein interactions. The review discusses key challenges and future prospects in the field, emphasiz-

ing the importance of an integrated multi-omics approach for the comprehensive micropeptidome mapping.

DOI: 10.1134/S0006297925602242

Keywords: micropeptides, small open reading frames, long non-coding RNAs, mass spectrometry, Ribo-Seq,

affinity labeling, co-immunoprecipitation

* To whom correspondence should be addressed.

INTRODUCTION

Any extended nucleotide sequence, whether nat-

ural or random, contains numerous open reading

frames (ORFs), as random nucleotide combinations

form various start and stop codons [1]. Historically,

large genome annotation consortia have focused ex-

clusively on identifying protein-coding ORFs and ig-

nored small ORFs (sORFs) encoding peptides shorter

than 100 amino acid residues [2]. For a long time,

sORFs had been considered an untranslated genomic

“noise,” incapable of encoding stable and functional

peptides.

This conception has changed dramatically with

the emergence of high-throughput sequencing tech-

nologies, particularly, ribosome profiling (Ribo-Seq),

which enables global mapping of translated RNA

regions at a single-nucleotide resolution [3]. Com-

bined with the proteomic data [4, 5], these studies

have provided compelling evidence for the trans-

lation of thousands of sORFs in various organisms,

including humans [6,  7]. Furthermore, function-

al studies have shown that many sORF translation

products – micropeptides – are involved in the reg-

ulation of fundamental processes, such as signaling,

metabolism, homeostasis, muscle activity, DNA repair,

and immune response [8-11]. Moreover, micropep-

tides have been found to influence the development

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BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

of various pathologies, including cancer. In other

words, micropeptides represent the “dark matter” of

the proteome and possess a significant regulatory po-

tential [12].

Due to some properties of micropeptides, includ-

ing their low cellular abundance, potential instabili-

ty, and difficulty in generating specific antibodies, the

search for these compounds and their characteriza-

tion are associated with methodological challenges,

which necessitated adaptation of existing methods

and development of new approaches for the reliable

identification, confirmation, and functional analysis

of micropeptides. The goal of this review is to provide

a comprehensive and systematic analysis of modern

methodological arsenal used in the studies of micro-

peptides and to discuss the prospects for new devel-

opments in this field.

MICROPEPTIDES

sORFs are short nucleotide sequences (no longer

than 100 codons from the start codon to the stop co-

don) in DNA or RNA. A significant number of sORFs

are located within protein-coding genes, in particu-

lar, in the 5′-untranslated regions (5′-UTRs) of mRNA

(the so-called upstream ORFs that often play a reg-

ulatory role in the translation of the main coding

sequence), 3′-untranslated regions (3′-UTRs) of mRNA

(downstream ORFs), or in the coding sequences in

alternative reading frames. Additionally, many sORFs

have been identified in transcripts initially classified

as non-coding RNAs, including long non-coding RNAs

(lncRNAs), primary microRNA transcripts, and circu-

lar RNAs [13].

sORFs can serve as templates for the synthesis of

short proteins, called micropeptides or microproteins.

Over two decades, numerous micropeptides involved

in embryogenesis [8,  9], metabolism [10,  11], and DNA

repair [14] have been described. Some micropeptides

are known to promote carcinogenesis, while others

act as tumor suppressors [15,  16]. Micropeptides with

the neuroprotective properties that inhibited the de-

velopment of the Alzheimer’s disease have also been

identified [17].

For example, physiologically active micropep-

tide DWORF functions as an activator of the calcium

ATPase SERCA responsible for transporting Ca

ions

from the cytoplasm into the sarcoplasmic reticulum (a

process necessary for muscle relaxation). Overexpres-

sion of DWORF in the cardiac muscle increases SERCA

activity, improves myocardial contractility, and affects

calcium homeostasis [18, 19]. Conversely, the micro-

peptides myoregulin (MLN), phospholamban (PLN),

and sarcolipin (SLN) inhibit SERCA and suppress its

activity [20, 21].

METHODS

FOR MICROPEPTIDE DISCOVERY

The search for and identification of function-

al micropeptides is a multi-step process that begins

with a large-scale screening of genome and tran-

scriptome for potential candidates. Historically, three

main approaches have been developed: bioinformatic

sequence analysis, global translation mapping by ri-

bosome profiling, and direct peptide detection using

mass spectrometry-based proteomics. In the last de-

cade, functional phenotypic screening has been added

to this list, allowing direct identification of micropep-

tides involved in specific cellular processes (Fig. 1).

BIOINFORMATIC APPROACHES

The bioinformatic search for sORFs is a non-triv-

ial task. The early methods of genome analysis used

to predict coding ORFs have set the minimal protein

product length as 100 amino acid residues. This re-

sulted in the loss of information about functional

peptides shorter than this threshold, despite known

existence of such molecules [22].

Firstly, the bioinformatic search for sORFs based

on the evolutionary sequence conservation across

different species [23]. An example of this approach

is the use of the Ka/Ks metric, which reflects the ra-

tio of non-synonymous to synonymous substitutions

in codons in the nucleotide sequence alignment for

different species and indicates whether the sequence

is under selective pressure [24]. More advanced meth-

ods have been developed later, e.g., PhyloCSF, which

uses codon substitution frequencies in both coding

and non-coding genomic regions [25].

To improve the reliability of predictions, other

features indicating the evolutionary conservation of

a sequence should be considered as well, such as the

absence of frameshift-causing insertions or deletions,

a decrease in the sequence conservatism at the edges

of coding regions, and others [26]. However, for short

sequences, the statistical significance of such analysis

is low [27]. Moreover, many sORFs are species-spe-

cific or arise de  novo in the genome, so a functional

peptide might not exhibit the sequence conservatism

and will be missed in analysis [6, 13].

The identification of sORFs is also possible

through the analysis of known features of coding se-

quences, such as their codon composition, GC content,

and others. One of the early algorithms involved iden-

tification of coding sequences by comparing the codon

frequencies in coding and non-coding genomic regions

[22, 28]. Another approach used analysis of six math-

ematical metrics of DNA sequence to assess the cod-

ing potential of sORFs [29]. Machine learning-based

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Fig.  1. Main approaches to the large-scale micropeptide discovery. Bioinformatic approaches include analysis of nu-

cleotide sequences to predict the coding potential of sORFs. The key methods are assessment of evolutionary conser-

vation across species, evaluation of sequence properties (e.g., codon composition), and application of machine learning

algorithms to integrate various features. Ribo-Seq (ribosome profiling) is an experimental method that maps all actively

translated regions of the transcriptome. It is based on ribosome stalling, followed by enzymatic cleavage of mRNA and

isolation and high-throughput sequencing of ribosome-protected fragments (footprints) in order to detect sORF transla-

tion in vivo. Mass spectrometry enables direct detection of sORF translation products and can be employed as either bot-

tom-up strategy, in which proteins are first digested by proteases into peptides before analysis, or top-down strategy

that implies analysis of intact micropeptides. Both strategies require careful sample preparation, including cell lysis, frac-

tionation for the enrichment with low-molecular-weight proteins, and analysis by liquid chromatography- tandem mass

spectrometry (LC-MS/MS). Phenotypic screenings aim to identify functional micropeptides. The loss-of-function screen-

ings use guide RNA libraries to knock out sORFs using CRISPR/Cas9, followed by the analysis of changes in the cell phe-

notype (e.g., cell proliferation rate). The gain-of-function screenings use sORF libraries to identify micropeptides whose

overexpression induces a specific cellular response.

analysis, which has become a common research tool

in recent years, allows identification of complex,

non-linear patterns in large datasets, as well as in-

tegration of various features of analyzed sequences,

including their evolutionary conservatism, predicted

structure, and other calculated metrics, to build high-

ly accurate predictive models [30, 31].

Most modern studies on the identification of cod-

ing sORFs use the same approaches for the analysis of

high-throughput sequencing data – ribosome profiling

(Ribo-Seq) and RNA sequencing (RNA-Seq), which sig-

nificantly increases the reliability of obtained results

[32, 33].

RIBOSOME PROFILING

High-throughput ribosome profiling was first

proposed in 2009 [3]. In the classic version of the

method, cells are treated with cycloheximide, which

binds to the E-site of the 60S ribosomal subunit and

inhibits translocation by stopping it at the translation

elongation stage [34]. The cells are then lysed and

treated with nucleases to cleave RNA, while regions

protected by stalled ribosomes are preserved. These

fragments, which are approximately 30 nucleotides

long, are called ribosome footprints. They are puri-

fied and sequenced using high-throughput methods.

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The resulting sequences are mapped onto the tran-

scriptome assembled based on the RNA-Seq data to

obtain a ribosome profile, i.e., the positions of ribo-

somes on mRNAs at the moment of translation ar-

rest with a single-nucleotide resolution [3]. The main

advantage of the approach is its ability to reflect the

translation levels of specific mRNAs at a given time,

thus allowing to study rapid changes in gene expres-

sion in response to external factors or during specific

cellular processes [35, 36].

Despite its broad capabilities, RNA-Seq has sever-

al limitations. In particular, the experimental results

depend significantly on the sample quality. The most

common problem is co-isolation of large ribonucleo-

protein complexes and non-coding RNAs along with

80S ribosomes, leading to false signals [37]. Also,

the percentage of footprint reads from the obtained

library is limited due to the contamination with ri-

bosomal RNA. Another fundamental problem is dis-

tortion of ribosome profiles because of the action of

certain antibiotics [38].

Other difficulties are associated with the analysis

of sORFs. Because of the sORF length, it is sometimes

impossible to accurately determine the translation ini-

tiation site if several potential start codons are located

nearby [36]. Another problem arises when sORFs over-

lap with the main ORF or are located within it [38].

To determine the exact position of start codons,

researchers use translation initiation inhibitors. These

compounds do not disrupt elongation or translation

termination, but stalls ribosomes mostly in the trans-

lation initiation regions, so ribosome profiles provide

accurate information about the position of ribosome

[20]. The first antibiotic used to map translation ini-

tiation sites was harringtonine [39] that binds to the

A-site of free 60S ribosomal subunit. After formation

of the 80S ribosome during translation initiation, har-

ringtonine blocks the transfer of methionine from the

initiator tRNA to the aminoacyl-tRNA located in the

A-site, leading to ribosome stalling at the translation

initiation site [40]. Importantly, harringtonine does not

bind to the 60S subunit in the content of the 80S ribo-

some and does not affect translation elongation and

termination; hence, the obtained footprints show po-

sitions of translation initiation sites [39, 40]. However,

when using harringtonine, some identified footprints

were found for the region located downstream of the

start codon, which, in certain cases, failed to provide

a sufficiently narrow peak for the initiation site map-

ping. A more accurate identification of start codons

can be achieved by using the GTI-Seq (Global Trans-

lation Initiation Sequencing) method, which employs

lactimidomycin to stall translation initiation. Lactimi-

domycin blocks the empty E-site of the 80S ribosome

and causes ribosome to stall immediately after trans-

lation initiation [41]. In a subsequent modification of

the method, QTI-Seq (Quantitative Translation Initia-

tion Sequencing), lysed cells are treated with lactim-

idomycin for a short period of time, followed by the

addition of puromycin to causes the dissociation of

elongating ribosomes. This approach allows to achieve

the maximum coverage of translation initiation sites,

while reducing the noise from elongating ribosomes

and artifacts associated with prolonged incubation of

cells with lactimidomycin [42].

Although ribosome profiling can identify translat-

ed regions, it cannot establish whether the identified

sORFs are coding, regulatory, or non-functional [43].

An indirect evidence of coding potential can be ob-

tained through bioinformatic analysis of candidate

sequences [44, 45]. The existence of an encoded pep-

tide in the cell can be proven by mass spectrometry

methods [46, 47].

MASS SPECTROMETRY

Mass spectrometry-based proteomic analysis al-

lows direct detection of sORF translation products.

As in classical proteomics, either peptide fragments

after digestion with proteases (bottom-up approach)

or intact proteins (top-down approach) can be ana-

lyzed. In both cases, identification of micropeptides

faces two main obstacles. First, it requires the use

of special sample preparation procedures to enrich

the sample with micropeptides because of their small

size, low stability, and low cellular abundance. Sec-

ond, identification of previously unannotated micro-

peptides requires the use of special reference data-

bases, as databases based on Ensembl, Ref-Seq, or

UniProt do not contain most potential micropeptides.

To prevent the degradation of micropeptides,

cell lysates are heated to 95°C to inactivate proteas-

es [5]. In some cases, this procedure is followed by

precipitation of large proteins with trichloroacetic

acid [48]. The resulting samples are fractionated by

polyacrylamide gel electrophoresis (PAGE) [49,  50],

size-exclusion chromatography [48], or reverse-phase

chromatography [51] to enriched them with low-mo-

lecular-weight proteins. More advanced methods of

preliminary fractionation have been developed, such

as GELFrEE (Gel-Eluted Liquid Fraction Entrapment

Electrophoresis, i.e., separation on a column contain-

ing polyacrylamide gel) [52, 53], ERLIC (Electrostatic

Repulsion Hydrophilic Interaction Chromatography,

i.e., chromatography based on electrostatic repulsion

and hydrophilic interaction) [54], and others. Depend-

ing on the selected approach, the proteins are then

either subjected to proteolysis (most often, with tryp-

sin) for the bottom-up analysis or directly analyzed by

liquid chromatography combined with tandem mass

spectrometry (LC-MS/MS).

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The identification of micropeptides is based on

matching experimental mass spectra with the theoret-

ical ones. However, construction of databases poses a

problem, as in  silico translation of all reading frames

in the genome or transcriptome covers almost all po-

tential micropeptides, but the size of the resulting da-

tabase is tens of times larger than that of standard

databases, leading to a high computational complex-

ity and high probability of false identifications [55].

Therefore, limited databases based on RNA-seq and

Ribo-Seq data are used [5,  56], which, however, does

not exclude the appearance of false-positive results.

In order to confirm an identified micropeptide its

isotopically labeled synthetic analog, which has an

identical mass spectrum that is shifted relative to the

spectrum of the validated peptide, is added in the re-

action [57, 58].

Micropeptides can be studied using the top-down

proteomics approach, which is particularly useful

for analyzing various proteoforms of micropeptides

formed by alternative splicing [59] and post-transla-

tional modifications [57]. For example, identification

of micropeptides presented by the major histocom-

patibility complex (MHC-I) allows to detect intact pep-

tides. A database of translated sequences obtained by

the ribosome profiling of cancer cells, was used to

identify thousands of previously unannotated pep-

tides by mass spectrometry [60].

PHENOTYPIC SCREENINGS

In addition to the analysis of sequences and

translation data, functional sORFs can be discovered

through phenotypic screening, which can be divided

into two approaches: loss-of-function screening and

gain-of-function screening.

CRISPR/Cas9 system has become the main tool for

the loss-of-function screening. In these experiments,

single-guide RNAs (sgRNAs) target Cas9 nuclease to

specific sORF regions due to a 20-nucleotide guide

sequence in the sgRNA and the presence of the pro-

tospacer adjacent motif (PAM) sequence immediate-

ly downstream of the targeted DNA region. Double-

strand breaks caused by Cas9 are repaired mostly

through the non-homologous end joining (NHEJ), lead-

ing to the insertions or deletions of nucleotides that

can cause frameshifts and appearance of premature

stop codons, thus preventing the biosynthesis of a

functional micropeptide [61].

Genome-wide and targeted screenings use the

sgRNA libraries targeting a large number of sORFs.

The libraries are typically delivered to the cells by

lentiviral vectors, allowing inactivation of thousands

of genomic loci in a single experiment [62]. Cells with

specific phenotypes resulting from the sORF knockout

are selected from the general population, and the cas-

settes containing the sgRNA sequences are sequenced

to identify the sORFs whose knockout caused chang-

es in the cell phenotype. Averaging the results across

multiple sgRNAs for a specific sORF allows to account

for the potential off-target effects of CRISPR/Cas9.

The phenotypes observed after CRISPR-mediated

inactivation of sORFs can range from changes in cell

growth, viability, morphology, signaling pathways, and

drug resistance to interactions with other molecules.

A specific phenotype used in the screening is chosen

in each particular experiment [62].

So far, CRISPR/Cas9-mediated phenotype-based

screenings have not yet been commonly accepted as

a method for identification of functional micropep-

tides. However, several functional micropeptides have

been discovered in proliferation screenings of librar-

ies based on Ribo-Seq data [63-65].

An alternative approach to the phenotype analysis

involves exogenous expression of constructs encoding

peptides from a pre-compiled library and cloned into

plasmid or lentiviral vectors [66]. The advantage of

this method is that it allows to analyze any sequence

without requiring the presence of specific motifs (as

in the case of CRISPR/Cas9). However, this approach

has the same drawbacks as the commonly used over-

expression of proteins for studying their functions.

CONFIRMATION OF EXISTENCE

AND CHARACTERIZATION OF MICROPEPTIDES

Although the methods for investigating micropep-

tides are similar to those used for conventional pro-

teins, the overall approaches to studying these classes

of molecules differ. Unlike “classical” proteins, most

micropeptides do not contain potential structural

domains, have a small size, and are present in cells

at low concentrations, which requires adjustment of

standard approaches. Moreover, if a micropeptide is

encoded in an lncRNA, it may be necessary to distin-

guish between the functional roles of the transcript

and the peptide encoded within it. The studies of

micropeptides include three main stages: candidate

selection, confirmation of its existence, and determi-

nation of its function.

There are two main approaches for the search

of potential candidates. The first is the analysis of

data obtained by high-throughput methods (see the

first part of the review). Typically, these methods

provide only indirect evidence of the micropeptide

existence, so they have to be supplemented by mass

spectrometry and Ribo-Seq data [46]. A completely

different approach is a manual search for sORFs and

annotation of the transcripts of interest. For exam-

ple, the micropeptide MIEF1-mp was found during the

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Fig.  2. Main strategies for studying micropeptides. The diagram shows key methods used to confirm the existence and

determine the structure, location, function, and molecular partners of a selected micropeptide candidate. The structure of

the micropeptide is determined by analysis of its secondary and tertiary structures. Thus, circular dichroism spectrosco-

py is used to assess elements of the secondary structure (α-helices, β-sheets). Nuclear magnetic resonance (NMR) allows

to determine the three-dimensional structure of small peptides in solution, while X-ray crystallography and cryo-elec-

tron microscopy are more often used to study the complexes of micropeptides with their partner proteins. Affinity la-

beling is a central approach in the studies of micropeptides. Fusing a micropeptide with short affinity tags (FLAG, HA,

6xHis) or fluorescent proteins (GFP, mCherry) allows its visualization in the cells by fluorescence microscopy, detection

by Western blotting, and isolation of protein complexes by co-immunoprecipitation (Co-IP). An alternative to affinity la-

beling is generation of specific antibodies to native micropeptides. The studies of micropeptide interactions includes sev-

eral approaches. Chemical labeling of synthetic micropeptides (e.g., with biotin) allows to search for the interacting pro-

teins in vitro in pull-down experiments, whereas weak or transient interactions in vivo can be investigated by proximity

labeling methods using BirA ligase (BioID) or ascorbate peroxidase (APEX), which covalently label neighboring proteins.

analysis of the 5′-UTR sequence of the MIEF1 gene

mRNA that encodes a described protein product [67].

Another example of this approach is the discovery of

the cancer-associated peptide PACMP in the analysis

of differential expression of lncRNAs in breast cancer

cells [68].

The second stage is confirmation of the micro-

peptide presence in the cells, as the protein product

of sORF translation may be unstable. In some cases,

it might be necessary to perform RACE (Rapid Am-

plification of cDNA Ends) to confirm the sequence of

the target transcript in the cell [69]. Direct evidence

of the micropeptide biosynthesis can be obtained by

mass spectrometry. For example, the presence of the

APPLE peptide was confirmed by mass spectrometry

after immunoprecipitation of the endogenously la-

beled peptide [46]. However, in most cases, the confir-

mation of translation of the studied sORF and biosyn-

thesis of a stable protein product can be achieved in

a simpler experiment by using exogenous expression

of the micropeptide fused with an affinity tag or a

fluorescent protein [70, 71].

The next step after confirmation of the micro-

peptide biosynthesis is elucidation of its function. The

methods used to study micropeptides are almost the

same as those used for conventional proteins and

include affinity labeling, Western blotting, immuno-

cytochemistry (ICC), proximity labeling, etc. (Fig.  2),

although most of these classical methods have to be

modified due to the small size of micropeptides.

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Fig.  3. The scheme of studies of the oncogenic micropeptide APPLE as an example of a comprehensive approach to mi-

cropeptide characterization. The diagram illustrates the three main stages of the study. I. Candidate selection: integrative

analysis of high-throughput Ribo-Seq, mass spectrometry, and RNA-Seq data led to the identification of the lncRNA ASH1L-AS1

as a potential source of the micropeptide. II. Confirmation of the micropeptide existence: APPLE biosynthesis in cells was

confirmed through two independent methods. (1)Exogenous expression: the coding sequence of APPLE was fused with var-

ious affinity and fluorescent tags (GFP, FLAG, HA), and the expression products were detected by fluorescence microscopy

and Western blotting. (2)Endogenous tagging: the FLAG tag was inserted into the native gene locus using the CRISPR/Cas9

genome editing system, which enabled isolation of the endogenous peptide via immunoprecipitation. III. Elucidation of

micropeptide function by functional analysis. (1) Studying the effect of peptide expression on the cell phenotype. Inactiva-

tion of APPLE expression led to cell apoptosis and proliferation arrest, while its overexpression promoted cell proliferation,

indicating the oncogenic role of APPLE. (2) Determination of APPLE intracellular location: immunofluorescence analysis re-

vealed that APPLE localizes to the endoplasmic reticulum. (3)Search for the APPLE protein partners. Co-IP followed by mass

spectrometry identified PABPC1 and eIF4G as micropeptide interacting partners. Subsequent studies confirmed that APPLE

is a part of the cap-binding complex and enhances translation initiation, thus promoting the development of malignancies.

The transcript of the lncRNA ASH1L-AS1 as a

potential source of the functional APPLE micropep-

tide, which promotes development of hematopoietic

malignancies, was first identified by integration of Ri-

bo-Seq, mass spectrometry, and RNA-Seq data (Fig.3).

The biosynthesis of APPLE was confirmed by multiple

methods, including generation of specific antibodies

against the micropeptide and endogenous tagging

with the CRISPR/Cas9 system, followed by detection

by Western blotting, immunoprecipitation, and mass

spectrometry analysis.

The functional role of APPLE in oncogenesis

was established using knockdown and overexpres-

sion experiments, which demonstrated its effects on

proliferation, differentiation, and apoptosis of acute

myeloid leukemia cells, both in  vitro and in in  vivo

(in a mouse model). A critical step in this study was

the phenotypic complementation experiment on the

background of the APPLE inactivation, which con-

firmed that the observed effects were specifical-

ly linked to the micropeptide rather than lncRNA

ASH1L-AS1 [46].

The information on the methods used to char-

acterize micropeptides is summarized in Online Re-

source 1.

KNOCKOUT AND KNOCKDOWN

To confirm the biological function of micropep-

tides, researchers can generate the cells in which the

corresponding encoding sequences are knocked-out

with genome editing systems (CRISPR/Cas9 or CRISPR-

Cas12a). Analysis of phenotypic changes in these cells

allows for assessing the role of investigated peptide.

However, in some cases, selecting guide RNAs may

be impossible due to the requirement for a PAM mo-

tif. The commonly used phenotypic markers are cell

proliferation rate, changes in the cell cycle, and oth-

ers [72]. An additional confirmation of the biological

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BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

significance of a micropeptide is the phenotype res-

toration through its exogenous expression in the

cells where this micropeptide had been the knocked

out [68].

A much less labor-intensive method for suppress-

ing expression of a target micropeptide is RNA in-

terference (RNAi)-mediated knockdown. Thus, small

interfering RNAs were used in the studies of micro-

peptides translated from lncRNAs [46, 73]. However,

in the case of sORFs encoded by mRNAs, RNAi-me-

diated knockdown will lead to the degradation of

the entire mRNA molecule, and consequently, the

observed effects may be associated with a decrease

in the expression of the main translation product of

this mRNA. In such cases, 2′-O-methyl antisense RNA

oligonucleotides complementary to the region around

the sORF start codon can be used to suppress the

biosynthesis of the micropeptide but not of the main

protein product [74].

AFFINITY TAGGING OF MICROPEPTIDES

Another important method in studying micropep-

tides is introduction of affinity tags. The small size of

micropeptides imposes restrictions on the choice of

the tag, as for many of them, the size of the intro-

duced sequence is comparable to the size of the pep-

tide itself. Moreover, the charge of the tag also plays

a significant role. A case of incorrect choice the tag

can disrupt the native localization and structure of the

micropeptide or its interaction with partner proteins.

The most common tags used with micropeptides

are small protein sequences, such as HA [75], FLAG

[11,  46], 6xHis [70]. In some studies, GFP or another

fluorescent protein are used. Fluorescent proteins can

be easily detected by microscopy and Western blotting

[10,  70], but there is a risk that the functions of the

tagged micropeptide in the cells can be disrupted.

Endogenous tagging by CRISPR/Cas9 is also used

to confirm the existence of micropeptides in the cells.

Insertion of affinity tag-encoding sequences directly

into the genomic locus allows for the detection of

micropeptides expressed from the native promoters,

which excludes artifacts associated with the overex-

pression of micropeptides and is a reliable method

for validating their biosynthesis.

The methods of affinity tagging of micropeptides

are discussed in detail in a recent review [76].

WESTERN BLOTTING

Western blotting is a standard method for detec-

tion and semi-quantitative determination of proteins.

However, the small size of micropeptides limits the

number of highly antigenic suitable epitopes, making

it difficult to generate antibodies against micropep-

tides. Nevertheless, for some peptides (over 50 amino

acids in length), the antibodies were successfully ob-

tained and used for Western blotting [70-78]. In most

cases, micropeptides are labeled with various affinity

tags (see above) for further detection [10, 70, 79]. It is

important to note that the content of native or endog-

enously tagged micropeptides in the cells can be very

low, making their detection extremely challenging.

Another limitation is the resolving power of

polyacrylamide gels used for the separation of pro-

teins from cell lysates. To increase the resolution of

low-molecular-weight proteins, researchers use the

Tris-tricine buffer system [80]. For example, it was

employed for the detection of the FLAG-tagged HOXB-

AS3 micropeptide (7  kDa) and native form of myoreg-

ulin (10  kDa) [71, 81]. However, small proteins weak-

ly bind to polyvinylidene fluoride and nitrocellulose

membranes used for the transfer and can be easily

lost during multiple membrane washes. In such cases,

alternative techniques can be applied, such as fixa-

tion on the membrane by crosslinking with blocking

proteins using formaldehyde or glutaraldehyde [82].

DETERMINATION OF MICROPEPTIDE

INTRACELLULAR LOCATION

The location of a micropeptide can help in elu-

cidating its protein partners and biological function.

A micropeptide located in the nucleus is likely to

interact with nuclear proteins, such as transcription

factors or chromatin-organizing proteins.

The most common method for determining the

location of micropeptides is immunocytochemistry,

which relies on the use of antibodies with a high spec-

ificity for the target antigen. The procedure typically

begins with cell fixation to preserve their morphology

and prevent degradation of cellular components. The

cells are then permeabilized to allow antibody access

into the cell. Generally, primary antibodies used in

the analysis are unlabeled and visualized using sec-

ondary antibodies conjugated with a fluorophore.

Similar to Western blotting, this method requires

antibodies against the target micropeptide, which can

be an obstacle. For example, antibodies against the

native micropeptide MP31 were used to determine its

mitochondrial location [83]. More often, immunocyto-

chemical staining is performed for the exogenously

expressed tagged micropeptide [45, 72, 79].

An alternative, widely used approach is the fu-

sion of micropeptides with fluorescent proteins (GFP,

mCherry, etc.), which allows their direct visualization

in the cells without the use of antibodies. However,

it may disrupt the properties of the micropeptide

METHODS FOR IDENTIFYING AND STUDYING MICROPEPTIDES 1529

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

because of the large size of the tag. To overcome this

limitation, split fluorescent proteins can be used, when

fluorescent protein, such as GFP, is divided into two

non-functional fragments – large fragment GFP1-10

and small GFP11, consisting of 16 amino acids and

used to tag the peptide of interest. The complementa-

tion of these fragments restores the tertiary structure

of the protein and its fluorescence [84, 85]. This system

was used to determine the location of the PIGBOS mi-

cropeptide, when the peptide tagged with three GFP11

repeats was co-expressed with GFP1-10 [77].

Chemical fluorescent tags have the least impact

on the native localization of micropeptides. However,

such experiments can be technically difficult due to

the need for the chemical synthesis of the micropep-

tide for its chemical labeling with a fluorescent ligand

(e.g., FITC). This labeling method was used to deter-

mine the location of the MP155 micropeptide [78].

The MicroID method is another tagging-based

method for identification of new micropeptides with

specific localization. This technique (a modification

of the BioID method, see below) uses biotin ligases

targeted to specific cellular compartments. After iso-

lation of covalent complexes, those containing ligase

crosslinked with low-molecular-weight proteins are

selected by fractionation and the bound micropep-

tides are identified by mass spectrometry [86].

IDENTIFICATION OF PROTEIN PARTNERS

Identification of protein partners of micropep-

tides is critically important for understanding the bi-

ological mechanisms involving these micropeptides.

The most widely used approach for studying protein–

protein interactions in  vivo is Co-IP, in which cells ex-

pressing a micropeptide labeled with an affinity tag

(most commonly, FLAG or HA) are lysed under mild

conditions that preserve existing protein complexes.

Antibodies specific to the affinity tag are added to

the cell lysate to bind the tagged micropeptide along

with all proteins that interact with it. The resulting

immune complexes are isolated from the solution,

usually with agarose or magnetic beads coated with

antibody-binding proteins  A or G. After thorough

washing to remove non-specifically bound proteins,

the captured complexes are eluted and identified,

most often by immunoprecipitation mass spectrome-

try (IP-MS) or Western blotting. This method was used

to demonstrate the interaction of the CYREN micro-

peptide with Ku70/80 and other proteins involved in

DNA repair [14]. The main requirement for Co-IP is a

sufficient stability of protein complexes to withstand

the lysis and washing procedures. However, the bind-

ing of a micropeptide to its protein partners is often

not strong enough.

The pull-down is a method similar to Co-IP, ex-

cept that the “bait” is typically a purified, tagged mi-

cropeptide immobilized on a solid phase. This can be

achieved through exogenous expression (e.g., with the

GST or 6xHis tag) or chemical synthesis, often with

the addition of the biotin tag. The immobilized bait is

incubated with the cell lysate or a solution of purified

proteins. Proteins that bind to the bait are retained

on the carrier. The carrier is washed, and the pro-

teins are then eluted and analyzed. For example, a

pull-down experiment with biotinylated P155 peptide

immobilized on streptavidin beads revealed the in-

teraction between this peptide and HSC70 chaperone

[78]. Unlike Co-IP, pull-down is convenient for con-

firming direct protein interactions in  vitro and does

not require specific antibodies. However, the results

may not take into account the influence of the cel-

lular context or the post-translational modifications

necessary for the interactions in  vivo.

To detect weaker or transient interactions in  vivo,

proximity labeling methods, such as BioID and APEX,

have been developed [87,  88]. These methods are

based on the expression of a chimeric protein con-

sisting of the target micropeptide fused with a special

enzyme. The enzyme is activated by the addition of

specific substrates that generate short-lived reactive

molecules (usually, biotin derivatives), which cova-

lently bind to proteins in a close proximity (within a

few nanometers) to the chimeric protein. The BioID

method uses the mutant biotin ligase BirA*, which,

in the presence of biotin and ATP, generates acti-

vated biotin-AMP that reacts with lysine residues of

neighboring proteins  [89]. In the APEX method, ascor-

bate peroxidase in the presence of biotin-phenol and

a short pulse of hydrogen peroxide rapidly (within

minutes) generates the biotin-phenoxyl radical that la-

bels tyrosine residues in a close proximity to the en-

zyme. After the labeling reaction, the cells are lysed,

biotinylated proteins are isolated using streptavidin

and identified by mass spectrometry [89]. The APEX

labeling method was used to search for the protein

partners of the mitochondrial peptide MIEF1 [79].

APEX labeling can also provide information on the

subcellular localization of micropeptides and their

functional environment, as demonstrated for C11orf98

in the nucleolus [90].

DETERMINATION

OF MICROPEPTIDE STRUCTURE

X-ray crystallography is rarely used for deter-

mining the three-dimensional structure of micro-

peptides, as it requires crystallization of proteins

or peptides into an ordered three-dimensional lat-

tice to obtain a diffraction pattern suitable for the

LAVROV et al.1530

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

structure resolution. Because of relatively small size,

micropeptides often do not form stable and well-or-

dered crystals suitable for obtaining high-resolution

diffraction patterns. Also, most micropeptides do

not have their own function but act as modulators

of protein partners and structural units of protein

complexes, so the crystal structure of the peptide

itself is often uninformative. However, X-ray crystal-

lography can be used to determine the structure of

peptide complexes with protein partners, for exam-

ple, the structure of phospholamban complex with

SERCA [91].

The presence of a stable secondary structure can

indicate the micropeptide stability in the cells and its

biological function. A relatively simple method for de-

termining the secondary structure of proteins is cir-

cular dichroism (CD) spectroscopy, which is based on

the differential absorption of circularly polarized light

by chiral molecules. Proteins, which are composed of

chiral amino acids, are optically active and exhibit

characteristic CD spectra. While this method does not

directly determine the tertiary structure, it is sensi-

tive to the presence of certain secondary structure

elements in the protein and can be used to elucidate

the types of secondary structures present in a micro-

peptide [75]. This approach was used to describe the

interaction of the CYREN peptide with the Ku70/Ku80

protein complex [14].

A more informative method for resolving the

tertiary structure of peptides is nuclear magnetic

resonance (NMR). Most micropeptides have small

molecular weights (up to 11 kDa) and, therefore, are

good subjects for the structure determination by NMR

[92]. This method was used for the characterization of

the DWORF structure and revealed a proline-induced

bend necessary for the activation of SERCA [19].

CONCLUSION

The studies of micropeptides encoded by sORFs

expand our understanding of the genome coding

potential and complexity of the proteome. Over the

past 10-15 years, a large body of evidence has been

accumulated regarding the functional role of micro-

peptides. As a result, sORFs are no longer considered

as non-functional sequences, as it has become clear

that they provide a new level in the regulation of

cellular processes. This paradigm shift has become

possible due to the prior development of molecular

biology methods. Ribosome profiling has revealed the

scale of sORF translation, while improvement in sam-

ple preparation for mass spectrometry allowed direct

detection of their peptide products, and CRISPR/Cas9-

based genome editing provided a powerful tool for

the functional studies.

Despite a significant progress, detection of micro-

peptides is still associated with certain challenges. It is

important to reliably differentiate sORFs that encode

micropeptides from those translated but non-function-

al or those that act as translational regulatory ele-

ments, which may require a deeper investigation of

their evolutionary origin and analysis by molecular

biology methods. The confirmation of the existence

of micropeptides and characterization of their prop-

erties remain a non-trivial task due to small size of

these molecules and limitations it imposes on the

use of classical molecular biology methods. Current

strategies used for micropeptide discovery may need

certain improvements. For example, the detection and

quantification of short-lived micropeptides require

further increase in the sensitivity of mass spectrom-

etry methods, optimization of sample preparation pro-

tocols, and generation of sufficiently comprehensive

but non-redundant reference databases.

Nevertheless, a substantial amount of data on mi-

cropeptides has already been accumulated. Future re-

search will likely involve more comprehensive integra-

tion of genomics, transcriptomics, and proteomics data.

This multi-omics approach will open new horizons in

studying the role of micropeptides and their potential

applications in disease diagnostics and therapy.

Abbreviations

Co-IP co-immunoprecipitation

GFP green fluorescent protein

Ribo-Seq ribosome profiling

RNA-seq RNA sequencing

sgRNA single-guide RNA

sORF small open reading frame

Supplementary information

The online version contains supplementary material

available at https://doi.org/10.1134/S0006297925602242.

Contributions

A.I.L. wrote the text of the article; O.A.D., M.P.R., and

N.M.Sh. edited the manuscript.

Funding

This work was supported by the Russian Science

Foundation (project no. 23-14-00058) and as part of

the State Assignment to the Lomonosov Moscow State

University (registration no.121031300037-7).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest

The authors of this work declare that they have

noconflicts of interest.

METHODS FOR IDENTIFYING AND STUDYING MICROPEPTIDES 1531

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

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