Proteins In Biological Samples Research Articles

Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography-Mass Spectrometry.

Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC-MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that "spacer peptides" can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC-tandem MS methods.

A panel of synthetic antibodies that selectively recognize and antagonize members of the interferon alpha family

The 12 distinct subtypes that comprise the interferon alpha (IFNα) family of cytokines possess anti-viral, anti-proliferative and immunomodulatory activities. They are implicated in the etiology and progression of many diseases, and also used as therapeutic agents for viral and oncologic disorders. However, a deeper understanding of their role in disease is limited by a lack of tools to evaluate single subtypes at the protein level. Antibodies that selectively inhibit single IFNα subtypes could enable interrogation of each protein in biological samples and could be used for characterization and treatment of disease. Using phage-displayed synthetic antibody libraries, we have conducted selections against 12 human IFNα subtypes to explore our ability to obtain fine-specificity antibodies that recognize and antagonize the biological signals induced by a single IFNα subtype. For the first time, we have isolated antibodies that specifically recognize individual IFNα subtypes (IFNα2a/b, IFNα6, IFNα8b and IFNα16) with high affinity that antagonize signaling. Our results show that highly specific antibodies capable of distinguishing between closely related cytokines can be isolated from synthetic libraries and can be used to characterize cytokine abundance and function.

Peroxyl radical- and photo-oxidation of glucose 6-phosphate dehydrogenase generates cross-links and functional changes via oxidation of tyrosine and tryptophan residues

Protein oxidation is a frequent event as a result of the high abundance of proteins in biological samples and the multiple processes that generate oxidants. The reactions that occur are complex and poorly understood, but can generate major structural and functional changes on proteins. Current data indicate that pathophysiological processes and multiple human diseases are associated with the accumulation of damaged proteins. In this study we investigated the mechanisms and consequences of exposure of the key metabolic enzyme glucose-6-phosphate dehydrogenase (G6PDH) to peroxyl radicals (ROO•) and singlet oxygen (1O2), with particular emphasis on the role of Trp and Tyr residues in protein cross-linking and fragmentation. Cross-links and high molecular mass aggregates were detected by SDS-PAGE and Western blotting using specific antibodies. Amino acid analysis has provided evidence for Trp and Tyr consumption and formation of oxygenated products (diols, peroxides, N-formylkynurenine, kynurenine) from Trp, and di-tyrosine (from Tyr). Mass spectrometric data obtained after trypsin-digestion in the presence of H216O and H218O, has allowed the mapping of specific cross-linked residues and their locations. These data indicate that specific Tyr-Trp and di-Tyr cross-links are formed from residues that are proximal and surface-accessible, and that the extent of Trp oxidation varies markedly between sites. Limited modification at other residues is also detected. These data indicate that Trp and Tyr residues are readily modified by ROO• and 1O2 with this giving products that impact significantly on protein structure and function. The formation of such cross-links may help rationalize the accumulation of damaged proteins in vivo.

Tissue Specific Labeling in Proteomics.

Mass spectrometry-based proteomics is a powerful tool for identifying and quantifying proteins in biological samples. While it is routinely used for the characterization of simple cell line systems, the analysis of the cell specific proteome in multicellular organisms and tissues poses a significant challenge. Isolating a subset of cells from tissues requires mechanical and biochemical separation or sorting, a process which can alter cellular signaling, and thus, the composition of the proteome. Recently, several approaches for cell selective labeling of proteins, that include bioorthogonal amino acids, biotinylating enzymes, and genetic tools, have been developed. These tools facilitate the selective labeling of proteins, their interactome, or of specific cell types within a tissue or an organism, while avoiding the difficult and contamination-prone biochemical separation of cells from the tissue. In this review, we give an overview of existing techniques and their application in cell culture models and whole animals.

Absolute venomics: Absolute quantification of intact venom proteins through elemental mass spectrometry

We report the application of a hybrid element and molecular MS configuration for the parallel absolute quantification of μHPLC-separated intact sulfur-containing venom proteins, via ICP triple quadrupole MS and 32S/34S isotope dilution analysis, and identification by ESI-QToF-MS of the toxins of the medically important African black-necked spitting cobra, Naja nigricollis (Tanzania); New Guinea small-eyed snake, Micropechis ikaheka; and Papuan black snake, Pseudechis papuanus. The main advantage of this approach is that only one generic sulfur-containing standard is required to quantify each and all intact Cys- and/or Met-containing toxins of the venom proteome. The results of absolute quantification are in reasonably good agreement with previously reported relative quantification of the most abundant protein families. However, both datasets depart in the quantification of the minor ones, showing a tendency for this set of proteins to be underestimated in standard peptide-centric venomics approaches. The molecular identity, specific toxic activity, and concentration in the venom, are the pillars on which the toxicovenomics-aimed discovery of the most medically-relevant venom toxins, e.g. those that need to be neutralized by an effective therapeutic antivenom, should be based. The pioneering venom proteome-wide absolute quantification shown in this paper represents thus a significant advance towards this goal. The potential of ICP triple quadrupole MS in proteomics in general, and venomics in particular, is critically discussed. Biological significanceAnimal venoms provide excellent model systems for investigating interactions between predators and prey, and the molecular mechanisms that contribute to adaptive protein evolution. On the other hand, numerous cases of snake bites occur yearly by encounters of humans and snakes in their shared natural environment. Snakebite envenoming is a serious global public health issue that affects the most impoverished and geopolitically disadvantaged rural communities in many tropical and subtropical countries. Unveiling the temporal and spatial patterns of venom variability is of fundamental importance to understand the molecular basis of envenoming, a prerequisite for developing therapeutic strategies against snakebite envenoming. Research on venoms has been continuously enhanced by advances in technology. The combined application of next-generation transcriptomic and venomic workflows has demonstrated unparalleled capabilities for venom characterization in unprecedented detail. However, mass spectrometry is not inherently quantitative, and this analytical limitation has sparked the development of methods to determine absolute abundance of proteins in biological samples. Here we show the potential of a hybrid element and molecular MS configuration for the parallel ESI-QToF-MS and ICP-QQQ detection and absolute quantification of intact sulfur-containing venom proteins via 32S/34S isotope dilution analysis. This configuration has been applied to quantify the toxins of the medically important African snake Naja nigricollis (Tanzania), and the Papuan species Micropechis ikaheka and Pseudechis papuanus.

Current trends in quantitative proteomics - an update.

Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large-scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd.

Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples

A method we previously developed has been applied to the determination of the glycosylation pattern of specific proteins in biological samples. Six proteins (alpha-1-antitrypsin, transferrin, haptoglobin, C1 inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the expected normal distribution of glycosylation patterns and to assess whether this methodology can be used to discriminate between samples from different individuals. For serum samples, the differences were shown to be small, while much larger differences were found for the CSF samples, with a greater number of glycoforms present. This can be linked to the occurrence of differential glycosylation in proteins expressed in the brain compared with proteins expressed elsewhere in the body. The developed method could distinguish differences in the glycosylation pattern of specific proteins in the individual samples, which was not reflected in the glycan content of total CSF. This is the first time that the glycoforms of several of these proteins have been investigated in CSF.

Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

Bead-Extractor Assisted Ready-to-Use Reagent System (BEARS) for Immunoprecipitation Coupled to MALDI-MS

Quantitative protein assays play an important role in the study of biological functions. Immunoassays and mass spectrometry are two main technologies for quantifying proteins in biological samples. The combination of immunoprecipitation (IP) with MALDI technology delivers high assay sensitivity and specificity, but the sample preparation procedure involves multiple washing and transfer steps. These steps can be performed either manually (requiring significant time and labor) or automatically (requiring the purchase of a complex liquid-handling workstation). This bottleneck has limited the widespread adoption of this technology. We present here the Bead-Extractor Assisted ready-to-use Reagent System (BEARS) technology for simplified, low cost protein and peptide immunoprecipitation combined with MALDI-MS detection. All of the reagents are stable during long-term storage and can be prepared in advance. In the BEARS technology, a magnetic-bead extractor is used to handle beads from 96 wells simultaneously. A BEARS-based method was developed for plasma renin activity (PRA) and was evaluated on fifty-three clinical samples. These experiments showed that the BEARS assay had an LOD and linear range comparable to the manual method and an automated iMALDI PRA assay, but was 4-times faster than the manual approach. The BEARS iMALDI results also correlated well with a conventional ELISA PRA assay, with a coefficient of determination of 0.98. The BEARS technology provides convenience and affordability, and extends the use of IP-based mass spectrometry technology to most research and clinical laboratories, including those in developing countries.

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

Surface-imprinted polymer coating l-cysteine-capped ZnS quantum dots for target protein specific recognition

This paper demonstrated a new method for preparing fluorescent molecularly imprinted polymer (MIP) for specific recognition of a target protein. The MIP-based fluorescent receptor was developed by coating MIP layer on the surface of l-cysteine modified Mn2+-doped ZnS quantum dots (QDs) using the surface molecular imprinting process. These MIP-QDs composites demonstrated fast adsorption kinetics, high stability, and good dispersibility in aqueous media. Since the fluorescence quenching of MIP-QDs composites is proportional to the concentration of the lysozyme, the MIP-based fluorescent receptor was successfully applied to the direct fluorescence quantification of lysozyme without further pretreatment. The MIP-based fluorescent receptor exhibited good selectivity and sensitivity for lysozyme detection. The optimum fluorescence intensity of the MIP-QDs was found to be at pH 6.0, and the linear range of lysozyme was from 0.1 to 2.0 μM with the detection limit of 25.2 nM. Moreover, the proposed fluorescent receptor was satisfactorily applied to the determination of lysozyme in real samples. This study provides a new approach for recognizing and detecting of specific proteins in biological samples.

Assessment of Inflammasome Activation by Cytokine and Danger Signal Detection.

The evaluation of the inflammasome activation usually addresses the presence of extracellular IL-1β and IL-18 or the secretion of danger signal proteins such as HMGB-1 through their quantification using an enzyme-linked immunosorbent assay (ELISA). The ELISA is a routine laboratory technique that uses antibodies and colorimetric changes to identify a substance of interest. ELISA uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid or wet sample. Using 96 well plates, the ELISA technique enables to quantify the concentration of a single cytokine in multiple samples. However, a limitation of IL-1β and IL-18 ELISA is the absence of discrimination between active and non-active form of the proteins, parameter critical, for example, to distinguish the biologically relevant IL-1β from its poorly active form pro-IL-1β. This issue can be solved using western blots or immunoblots (IB), a common analytical procedure to detect the presence of different proteins in biological samples. Using denaturating conditions, IB allows the visualization of different sizes of the proteins of choice and is a commonly used technique in the inflammasome field to evaluate, for instance, the maturation of pro-IL-1β, pro-IL-18, and pro-caspase-1 into mature IL-1β, mature IL-18, and mature caspase-1, respectively. Moreover inflammasome activation may lead to the release of inflammasome particles outside the cell through caspase-1- or caspase-11-dependent cell death mechanism termed pyroptosis. In this case, NLR, ASC, and caspase-1 components are detectable outside the cell using IB analysis. ELISA and IB can be performed on cell culture supernatant or cell extract and on ex vivo samples from organ homogenates or biological fluids such as serum and plasma or bronchoalveolar lavages.

Mutant Proteogenomics.

Identification of mutant proteins in biological samples is one of the emerging areas of proteogenomics. Despite the fact that only a limited number of studies have been published up to now, it has the potential to recognize novel disease biomarkers that have unique structure and desirably high specificity. Such properties would identify mutant proteoforms related to diseases as optimal drug targets useful for future therapeutic strategies. While mass spectrometry has demonstrated its outstanding analytical power in proteomics, the most frequently applied bottom-up strategy is not suitable for the detection of mutant proteins if only databases with consensus sequences are searched. It is likely that many unassigned tandem mass spectra of tryptic peptides originate from single amino acid variants (SAAVs). To address this problem, a couple of protein databases have been constructed that include canonical and SAAV sequences, allowing for the observation of mutant proteoforms in mass spectral data for the first time. Since the resulting large search space may compromise the probability of identifications, a novel concept was proposed that included identification as well as verification strategies. Together with transcriptome based approaches, targeted proteomics appears to be a suitable method for the verification of initial identifications in databases and can also provide quantitative insights to expression profiles, which often reflect disease progression. Important applications in the field of mutant proteoform identification have already highlighted novel biomarkers in large-scale investigations.

Quantitative Analysis of Tissue Samples by Combining iTRAQ Isobaric Labeling with Selected/Multiple Reaction Monitoring (SRM/MRM).

Mass spectrometry-based phosphoproteomics is an indispensible technique used in the discovery and quantification of phosphorylation events on proteins in biological samples. The application of this technique to tissue samples is especially useful for the discovery of biomarkers as well as biological studies. We herein describe the application of a large-scale phosphoproteome analysis and SRM/MRM-based quantitation to develop a strategy for the systematic discovery and validation of biomarkers using tissue samples.

Peptide identification in “shotgun” proteomics using tandem mass spectrometry: Comparison of search engine algorithms

High-throughput proteomics technologies are gaining popularity in different areas of life sciences. One of the main objectives of proteomics is characterization of the proteins in biological samples using liquid chromatography/mass spectrometry analysis of the corresponding proteolytic peptide mixtures. Both the complexity and the scale of experimental data obtained even from a single experimental run require specialized bioinformatic tools for automated data mining. One of the most important tools is a so-called proteomics search engine used for identification of proteins present in a sample by comparing experimental and theoretical tandem mass spectra. The latter are generated for the proteolytic peptides derived from a protein database. Peptide identifications obtained with the search engine are then scored according to the probability of a correct peptide-spectrum match. The purpose of this work was to perform a comparison of different search algorithms using data acquired for complex protein mixtures, including both annotated protein standards and clinical samples. The comparison was performed for three popular search engines: commercially available Mascot, as well as open-source X!Tandem and OMSSA. It was shown that the search engine OMSSA identifies in general a smaller number of proteins, while X!Tandem and Mascot deliver similar performance. We found no compelling reasons for using the commercial search engine instead of its open source competitor.

Aptamer-based detection and quantitative analysis of human immunoglobulin E in capillary electrophoresis with chemiluminescence detection.

A novel aptamer-based CE with chemiluminescence (CL) assay was developed for highly sensitive detection of human immunoglobulin E (IgE). The IgE aptamer was conjugated with gold nanoparticles (AuNPs) to form AuNPs-aptamer that could specifically recognize the IgE to produce an AuNPs-aptamer-IgE complex. The mixture of the AuNPs-aptamer-IgE complex and the unbounded AuNPs-aptamer could be effectively separated by CE and sensitively detected with luminol-H2 O2 CL system. By taking the advantage of the excellent catalytic behavior of AuNPs on luminol-H2 O2 CL system, the ultrasensitive detection of IgE was achieved. The detection limit of IgE is 7.6 fM (S/N = 3) with a linear range from 0.025 to 250 pM. Successful detection of IgE in human serum samples was demonstrated and the recoveries of 94.9-103.2% were obtained. The excellent assay features of the developed approach are its specificity, sensitivity, adaptability, and very small sample consumption. Our design provides a methodology model for determination of rare proteins in biological samples.

Attomolar detection of proteins via cascade strand-displacement amplification and polystyrene nanoparticle enhancement in fluorescence polarization aptasensors.

Extremely sensitive and accurate measurements of protein markers for early detection and monitoring of diseases pose a formidable challenge. Herein, we develop a new type of amplified fluorescence polarization (FP) aptasensor based on allostery-triggered cascade strand-displacement amplification (CSDA) and polystyrene nanoparticle (PS NP) enhancement for ultrasensitive detection of proteins. The assay system consists of a fluorescent dye-labeled aptamer hairpin probe and a PS NP-modified DNA duplex (assistant DNA/trigger DNA duplex) probe with a single-stranded part and DNA polymerase. Two probes coexist stably in the absence of target, and the dye exhibits relatively low FP background. Upon recognition and binding with a target protein, the stem of the aptamer hairpin probe is opened, after which the opened hairpin probe hybridizes with the single-stranded part in the PS NP-modified DNA duplex probe and triggers the CSDA reaction through the polymerase-catalyzed recycling of both target protein and trigger DNA. Throughout this CSDA process, numerous massive dyes are assembled onto PS NPs, which results in a substantial FP increase that provides a readout signal for the amplified sensing process. Our newly proposed amplified FP aptasensor enables the quantitative measurement of proteins with the detection limit in attomolar range, which is about 6 orders of magnitude lower than that of traditional homogeneous aptasensors. Moreover, this sensing method also exhibits high specificity for target proteins and can be performed in homogeneous solutions. In addition, the suitability of this method for the quantification of target protein in biological samples has also been shown. Considering these distinct advantages, the proposed sensing method can be expected to provide an ultrasensitive platform for the analysis of various types of target molecules.

Dual-signal amplification strategy for ultrasensitive chemiluminescence detection of PDGF-BB in capillary electrophoresis.

Many efforts have been made toward the achievement of high sensitivity in capillary electrophoresis coupled with chemiluminescence detection (CE-CL). This work describes a novel dual-signal amplification strategy for highly specific and ultrasensitive CL detection of human platelet-derived growth factor-BB (PDGF-BB) using both aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (HRP-AuNPs-aptamer) as nanoprobes in CE. Both AuNPs and HRP in the nanoprobes could amplify the CL signals in the luminol-H2 O2 CL system, owing to the excellent catalytic behavior of AuNPs and HRP in the CL system. Meanwhile, the high affinity of aptamer modified on the AuNPs allows detection with high specificity. As proof-of-concept, the proposed method was employed to quantify the concentration of PDGF-BB from 0.50 to 250 fm with a detection limit of 0.21 fm. The applicability of the assay was further demonstrated in the analysis of PDGF-BB in human serum samples with acceptable accuracy and reliability. The result of this study exhibits distinct advantages, such as high sensitivity, good specificity, simplicity, and very small sample consumption. The good performances of the proposed strategy provide a powerful avenue for ultrasensitive detection of rare proteins in biological sample, showing great promise in biochemical analysis.

A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS)

Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with 34S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and 34S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (msp/msam) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5–3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations.

Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples.

Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.