Use of HIC high resolution chromatography and elastase for bottom up proteomics

One of the key applications used to characterize single or complex protein mixtures via bottom up proteomics is liquid chromatography−tandem mass spectrometry (LC−MS/MS).
Recent technical advances allow for identification of >10 000 proteins in a cancer cell line. On the peptide level chromatography methods, like strong cation exchange (SCX)
and hydrophilic interaction chromatography (HILIC), as well as high-pH reversed phase chromatography have been employed successfully. Because of its robustness
and ease of handling, the classical and still widely used approach for protein fractionation prior to LC− MS/MS is gel-based separation under denaturing conditions (SDS-PAGE).
Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used
to study post-translational modifications of proteins and drug−protein interactions.  HIC is a high-resolution chromatography mode based on the interaction of
weakly hydrophobic ligands of the stationary phase with hydrophobic patches on the surface of the tertiary structure of proteins. By employment of high concentrations
of structure-promoting (“kosmotropic”) salts, proteins in HIC retain their conform

In a recent publication, HIC was used to separate proteins, followed by bottom up LC−MS/MS experiments (1).  HIC was used to fractionate antibody species
followed by comprehensive peptide mapping as well as to study protein complexes in human cells. The results indicated that HIC−reversed-phase chromatography (RPC)
mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

An additional observation noted that tryptic digests of the antibody used in the study yielded a protein coverage of 56% for the light chain and 63.2% for the
heavy chain. A consecutive proteolytic digestion protocol combing on-filter trypsin and elastase digestion drastically improved sequence coverage of
both light (100%) and heavy chains (99.2%).

1. Rackiewicz, M. et al. (2017) Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes. J.Proteome.Res. 16, 2318–23.

Why wait ? Sample prep/protein digestion in as little as 30 minutes!

While many proteases are used in bottom-up mass spectrometric (MS) analysis, trypsin (4,5) is the de facto protease of choice for most applications. There are several reasons for this: Trypsin is highly efficient, active and specific. Tryptic peptides produced after proteolysis are ideally suited, in terms of both size (350–1,600 Daltons) and charge (+2 to +4), for MS analysis. One significant drawback to trypsin digestion is the long sample preparation times, which typically range from 4 hours to overnight for most protocols. Achieving efficient digestion usually requires that protein substrates first be unfolded either with surfactants or denaturants such as urea or guanidine. These chemical additives can have negative effects, including protein modification, inhibition of trypsin or incompatibility with downstream LC-MS/MS. Accordingly, additional steps are typically required to remove these compounds prior to analysis.

To shorten the time required to prepare samples for LC-MS/MS analysis, we have developed a specialized trypsin preparation that supports rapid and efficient digestion at temperatures as high as 70°C. There are several benefits to this approach. First, proteolytic reaction times are dramatically shortened. Second, because no chemical denaturants have been added, off -line sample cleanup is not necessary, leading to shorter preparation times and diminished sample losses.

The Rapid Digestion trypsin protocols are highly flexible. They can accommodate a variety of additives including reducing and alkylating agents. There are no restrictions on sample volume or substrate concentrations with these kits. Furthermore, the protocol is simple to follow and requires no laboratory equipment beyond a heat block. Digestion is achieved completely using an in-solution approach, and since the enzyme is not immobilized on beads, the protocol does not have strict requirements for rapid shaking and off -line filtering to remove beads.

In addition to the benefits of this flexibility, we also developed a Rapid Digestion–Trypsin/Lys-C mixture. Like the Trypsin/Lys-C Mix previously developed to prepare maximally efficiently proteolytic digests, particularly for complex mixtures, Rapid Digestion–Trypsin/Lys C is ideally suited for studies that require improved reproducibility across samples.


Making BRET the Bright Choice for In vivo Imaging: Use of NanoLuc® Luciferase with Fluorescent Protein Acceptors

13305818-cr-da-nanoluc-application_ligundLive animal in vivo imaging is a common and useful tool for research, but current tools could be better. Two recent papers discuss adaptations of BRET technology combining the brightness of fluorescence with the low background of a bioluminescence reaction to create enhanced in vivo imaging capabilities.

The key is to image photons at wavelengths above 600nm, as lower wavelengths are absorbed by heme-containing proteins (Chu, J., et al., 2016 ). Fluorescent protein use in vivo is limited because the proteins must be excited by an external light source, which generates autofluorescence and has limited penetration due to absorption by tissues. Bioluminescence imaging continues to be a solution, especially firefly luciferase (612nm emission at 37°C), but its use typically requires long image acquisition times. Other luciferases, like NanoLuc, Renilla, and Gaussia, etc. either do not produce enough light or the wavelengths are readily absorbed by tissues, limiting their use to near- surface imaging.

The two papers discussed here illustrate how researchers have combined NanoLuc® luciferase with a fluorescent protein to harness bioluminescent resonance energy transfer (BRET) for brighter in vivo imaging reporters. Continue reading

Widening the Proteolysis Bottleneck: A New Protein Sample Preparation Tool

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

Improvements in Protein Bioprocessing

As more and more protein-based therapeutics enter research pipelines, more efficient protocols are needed for characterization of protein structure and function, as well as means of quantitation. One main step in this pipeline, proteolysis of these proteins into peptides, presents a bottleneck and can require optimization of multiple steps including reduction, alkylation and digestion time.

We have developed a new trypsin reagent, Rapid Digestion–Trypsin, that streamlines the protein sample preparation process, reducing the time to achieve proteolysis to about 1 hour, a remarkable improvement over existing overnight sample preparation times.

How Does it Work?

With this new trypsin product, proteolysis is performed at 70°C, incorporating both denaturation and rapid digestion. The protocol can be used with multiple protein types, including pure proteins and complex mixtures, and is compatible with digestion under native, reduced or nonreduced conditions.

Continue reading

Improved Method for the Rapid Analysis of Monoclonal Antibodies Using IdeS

ides_abTherapeutic monoclonal antibodies (MAbs) are inherently heterogeneous due to a wide range of both enzymatic and chemical modifications, such as oxidation, deamidation and glycosylation which may occur during expression, purification or storage. For identification and functional evaluation of these modifications, stability studies
are typically performed by employing stress conditions such as exposure to chemical oxidizers, elevated pH and temperature.

To characterize MAbs, a variety of analytical techniques are chosen, such as size exclusion chromatography and ion exchange chromatography. However, due to the large size of the intact MAbs, these methods lack structural resolution. Often, the chromatographic peaks resolved by SEC and IEC methods are collected and further analyzed by peptide mapping to obtain more detailed information. Peptide mapping, in which antibodies are cleaved into small peptides through protease digestion followed by LC–MS/MS analysis, is generally the method of choice for detection and quantitation of site-specific modifications. However sample preparation and lengthy chromatographic separation make peptide mapping impractical for the analysis of large numbers of samples. In contrast to peptide mapping analysis, the middle-down approach offers the advantage of high-throughput and specificity for antibody characterization.

Limited proteolysis of IgG molecules by the IdeS enzyme has been introduced for antibody characterization due to its high cleavage specificity and simple digestion procedure. Continue reading

Protein:DNA Interactions—High-Throughput Analysis

Protein-DNA interactions are fundamental processes in gene regulation in a living cells. These interactions affect a wide variety of cellular processes including DNA replication, repair, and recombination. In vivo methods such as chromatin immunoprecipitation (1) and in vitro electrophoretic mobility shift assays (2) have been used for several years in the characterization of protein-DNA interactions. However, these methods lack the throughput required for answering genome-wide questions and do not measure absolute binding affinities. To address these issues a recent publication (3) presented a high-throughput micro fluidic platform for Quantitative Protein Interaction with DNA (QPID). QPID is an microfluidic-based assay that cam perform up to 4096 parallel measurements on a single device.

The basic elements of each experiment includes oligonucleotides that were synthesized and hybridized to a Cy5-labeled primer and extended using Klenow. All transcription factors that were evaluated contained a 3’HIS and 5’ cMyc tag and were expressed in rabbit reticulocyte coupled transcription and translation reaction (TNT® Promega). Expressed proteins are loaded onto to the QIPD device and immobilized. In the DNA binding assay the fluorescent DNA oligonucleotides are incubated with the immobilized transcription factors and fluorescent images taken. To validate this concept the binding of four different transcription factor complexes to 32 oligonucleotides at 32 different concentrations was characterized in a single experiment. In a second application, the binding of ATF1 and ATF3 to 128 different DNA sequences at different concentrations were analyzed on a single device.

Literature Cited

  1. Ren, B. et al. (2007) Genome-wide mapping of in vivo protein-DNA binding proteins. Science 316, 1497–502.
  2. Garner, M.M. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions. Nuc. Acids. Res. 9, 3047-60.
  3. Glick,Y et al. (2016) Integrated microfluidic approach for quantitative high throughput measurements of transcription factor binding affinities. Nuc. Acid Res. 44, e51.

Optimization of Alternative Proteases for Bottom-Up Proteomics

Alternate Proteases CoverBottom-up proteomics focuses on the analysis of protein mixtures after enzymatic digestion of the proteins into peptides. The resulting complex mixture of peptides is analyzed by reverse-phase liquid chromatography (RP-LC) coupled to tandem mass spectrometry (MS/MS). Identification of peptides and subsequently proteins is completed by matching peptide fragment ion spectra to theoretical spectra generated from protein databases.

Trypsin has become the gold standard for protein digestion to peptides for shotgun proteomics. Trypsin is a serine protease. It cleaves proteins into peptides with an average size of 700-1500 daltons, which is in the ideal range for MS (1). It is highly specific, cutting at the carboxyl side of arginine and lysine residues. The C-terminal arginine and lysine peptides are charged, making them detectable by MS. Trypsin is highly active and tolerant of many additives.

Even with these technical features, the use of trypsin in bottom-up proteomics may impose certain limits in the ability to grasp the full proteome, Tightly-folded proteins can resist trypsin digestion. Post-translational modifications (PTMs) present a different challenge for trypsin because glycans often limit trypsin access to cleavage sites, and acetylation makes lysine and arginine residues resistant to trypsin digestion.

To overcome these problems, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated when using these alternative proteases have not been systematically documented.

In a recent reference (2), optimized protocols for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, Lys-C, Lys-N, Asp-N, Glu-C and Arg-C have been created.

Data describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in approximately in 2 days.


  1. Laskay, U. et al. (2013) Proteome Digestion Specificity Analysis for the Rational Design of Extended Bottom-up and middle-down proteomics experiments. J of Proteome Res. 12, 5558–69.
  2. Giansanti, P. et. al. (2016) Six alternative protease for mass spectrometry based proteomics beyond trypsin. Nat. Protocols 11, 993–6

High-Throughput Screening for Potential Biomarkers Using Cerebrospinal Fluid (CSF)

3240CA02_1A_rename_3Cerebrospinal fluid (CSF) is a bodily fluid present around the brain and in the spinal cord. It acts as a protective cushion against shocks and participates in the immune response in the brain. Analysis of total CSF protein can be used for diagnostic purposes, as, for instance, a sign of a tumor, bleeding, inflammation, or injury. Considering the high value of CSF as a source of potential biomarkers for brain-associated damages and pathologies, the development of robust automated platform for CSF proteomics is of great value.

The scalable automated proteomic pipeline (ASAP2)  was initially developed with the purpose of (i) discovering protein biomarkers in plasma (1). A summary of the ASAP2 process is as follows:As a first step, abundant-protein immuno-affinity depletion is performed with antibody-based columns and LC systems equipped with a refrigerated autosampler and fraction collector. This block is linked to and followed by buffer exchange performed in a 96-well plate format by manual operations that require <1 h to be completed. The rest of the process is fully automated and includes (i) reduction, alkylation, enzymatic digestion.; (ii) tandem mass tag (TMT) labeling and pooling (processing time of ); (iii) RP solid-phase extraction (SPE) purification ; and (iv) strong cation-exchange (SCX) SPE purification.

A recent reference (2) validated the use of ASAP2 for sample preparation and proteomic analysis of human CSF samples was performed. CSF samples were first depleted from abundant proteins by multiplexed immuno-affinity. Subsequently, reduction, alkylation, protein digestion (using Trypsin/Lys-C), TMT 6-plex labeling, pooling, and sample cleanup were performed in a 96-well-plate format using a liquid-handling robotic platform. Ninety-six  identical CSF samples were prepared using the highly automated ASAP2 procedure. Proteome coverage consistency, quantitative precision, and individual protein variability, were determined. Results indicated that, ASAP2 is efficient in analyzing large numbers of human CSF samples and would be a valuable tool for biomarker discovery.


  1. Dayon, L et al. (2014) Comprehensive and Scalable Highly Automated MS-Based Proteomic Workflow for Clinical Biomarker Discovery in Human Plasma. J of Proteome Res. 13, 3837–45
  2. Galindo, M-N. et al. (2015) Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery. Anan. Chem. 87, 10755–61

Dino Protein: New Methods for Old (Very) Samples

Hadrosaurus skeleton vintage engraving.

Hadrosaurus skeleton vintage engraving.

Brachylophosaurus was a mid-sized member of the hadrosaurid family of dinosaurs living about 78 million years ago, and is known from several skeletons and bonebed material from the Judith River Formation of Montana and the Oldman Formation of Alberta. Recent fossil evidence indicates structures similar to blood vessels in location and morphology, have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty  network after degradation of original organic components (i.e., bacterial, slime mold or fungal in origin).  Cleland et al. (1) tested the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence. Continue reading

ProteaseMAX: A Surfactant for the Most Complex Mixtures

Alternate Proteases CoverHere we provide two examples of “atypical” experiments that take advantage of the properties of the ProteaseMAX™ Surfactant to improve studies involving digestion of complex protein mixtures.

Example 1
Clostridium difficile spores are considered the morphotype of infection, transmission and persistence of C. difficile infections. A recent publication (1) illustrated a novel strategy using three different approaches  to identify proteins of the exosporium layer of C. difficile spores and complements previous proteomic studies on the entire C. difficile spores. Continue reading